Genetically engineered Vibrio sp. and uses thereof

ABSTRACT

The present disclosure relates to the seminal discovery of a generation and use of genetically engineered Vibrio sp. Provided is the use of the genetically engineered bacteria for the construction, maintenance, manipulation, and/or propagation of DNA constructs; protein expression; protein secretion; vectors and other metabolic tools; metabolic engineering; expression of cellular extracts for cell-free biology; shuttle vectors; cloning vectors; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. as a cloning or expression vector for replication of recombinant DNA constructs. The disclosure also relates to methods of use of the above.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/367,106 filed Dec. 1, 2016, now issued as U.S. Pat. No.10,377,997; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 62/261,758 filed Dec. 1, 2015. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference into the application. The accompanying sequence listingtext file, named DNASGI1910-2_ST25.txt, was created on Jul. 26, 2019 andis 42 KB in size. The file can be accessed using Microsoft Word on acomputer that uses Window OS.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure relates generally to genetically engineeredVibrio sp. bacteria and the use of such bacteria for the construction,maintenance, manipulation, and/or propagation of recombinant DNA, andprotein expression.

Background Information

The biotechnology sector relies upon organisms such as E. coli as hostsfor the generation of desired biomolecules (e.g., recombinant DNA,proteins, natural products, etc.) as well as for the study of biologicalprocesses and the development of bio-based technologies and products.While advances in fields such as genomics, synthetic biology, andgenome/metabolic engineering have made possible projects at anunprecedented scale, the host organisms that the field relies upon havechanged relatively little in decades and are proving to be inadequate orinefficient for many ambitious projects.

E. coli has been the main prokaryotic workhorse for several decades,being used ubiquitously in both academic and industrial efforts, andrelied upon as a host for molecular cloning, protein expression,metabolic engineering, a source of cellular extracts for in vitromolecular biology, and as a chassis for synthetic biology efforts. Theuse of E. coli is due largely to its extensive characterization (havingserved as a model organism since the late 19th century), having a largecollection of standardized tools and protocols, and being relativelyeasy to work with. E. coli is certainly not the only organism in use inbiotechnology, as there are plenty of obscure organisms being utilized,usually to leverage some peculiar biological property that allows thatorganism to excel in some niche application, but E. coli is hands downthe most widely adopted and broadly applied bacterial species inbiotechnology.

There is a need for robust, faster growing, and easily geneticallymanipulated bacterial cells that can be used as host organisms,especially to produce products such as large recombinant DNA molecules,and as alternative hosts for protein and peptide expression.

SUMMARY OF THE INVENTION

The present disclosure relates to the generation of geneticallyengineered Vibrio sp. bacteria. Specifically, the disclosure relates tothe use of the genetically engineered bacteria for the construction,maintenance, manipulation, and/or propagation of DNA constructs; proteinexpression; metabolic engineering; expression of cellular extracts forcell-free biology; and for synthetic biology applications. Thedisclosure also relates to the use of the replication machinery ofVibrio sp. on a cloning vector for replication of recombinant DNAconstructs.

In some aspects, the present disclosure provides a geneticallyengineered Vibrio sp. bacteria comprising an altered Chromosome I orChromosome II. In some examples, one or more non-essential genes areremoved from either Chromosome I or Chromosome II. In some examples, theone or more removed genes encode an element selected from the groupconsisting of an endonuclease, an exonuclease, a methylase, a nuclease,a restriction enzyme, and a restriction-modification system.

In some aspects, the present disclosure provides a geneticallyengineered Vibrio sp. bacteria, wherein at least one essential elementfrom Chromosome II is alternatively located on an engineered ChromosomeI. In some examples, the essential element is a gene required for afunction selected from the group consisting of metabolism, DNAreplication, transcription, translation, cellular structure maintenance,and transport processes into or out of the cell.

In some aspects, the present disclosure provides a geneticallyengineered Vibrio sp. bacteria that contains only one chromosome. Insome examples, the single chromosome comprises essential elements fromChromosome I and Chromosome II such that the single chromosome iscapable of supporting survival and replication of the bacteria undernon-selective conditions.

In some aspects, the herein disclosed genetically engineered Vibrio sp.further comprises a heterologous nucleic acid sequence operably linkedto a heterologous promoter. In some examples the heterologous nucleicacid encodes T7 RNA polymerase. In some examples, the heterologouspromoter is an inducible promoter.

In some aspects, the present disclosure provides a process for producingcompetent Vibrio sp. cells comprising: (a) growing Vibrio sp. cells in agrowth-conducive medium; (b) rendering said Vibrio sp. cells competent;and (c) freezing the cells. In some examples, the Vibrio sp. bacterialcells are any of those disclosed herein. In some examples, rendering thecells competent comprises growing the cells in a solution withsupplemented salts.

In some aspects, the present disclosure provides a method of producing abiomolecule comprising: a) contacting Vibrio sp. bacteria with aheterologous nucleic acid encoding the biomolecule, such that theheterologous nucleic acid is introduced into the bacteria; b) growingthe bacteria in a growth-conducive medium wherein the heterologousnucleic acid is expressed, thereby producing the biomolecule; and c)isolating the biomolecule. In some examples, the method is performedusing any of the bacteria disclosed herein. In some examples, theheterologous nucleic acid comprises a nucleic acid sequence encodingVibrio sp. replication machinery. In some examples, the replicationmachinery comprises SEQ ID NO: 1. In some examples, the heterologousnucleic acid further comprises an inducible promoter operably linked tothe nucleic acid encoding the biomolecule. In some examples, the Vibriosp. bacteria are naturally competent. In some examples, the Vibrio sp.bacteria are competent cells generated by any of the methods disclosedherein. In some examples, the nucleic acid is introduced by conjugation,chemical competence, natural competence, or electroporation. In someexamples, the herein disclosed method further comprises monitoring thegrowth conducive media for the presence of the biomolecule over time.

In some aspects, the present disclosure provides an isolated orsynthesized nucleic acid molecule comprising SEQ ID NO:1. In someexamples, the isolated or synthesized nucleic acid molecule furthercomprises heterologous sequence on the 5′ and 3′ end, wherein theheterologous 5′ and 3′ sequences are compatible for cloning into atarget vector.

In some aspects, the present disclosure provides a vector comprisingVibrio sp. chromosomal replication machinery. In some examples, thereplication machinery comprises SEQ ID NO: 1. In some examples, thevector further comprises a heterologous nucleic acid of interest. Insome examples, the vector further comprises an inducible promoteroperably linked to a nucleic acid of interest. In some examples, thevector is capable of replication in E. coli or S. cerevisiae.

In some aspects, the present disclosure provides a compositioncomprising any of the genetically engineered Vibrio sp. disclosedherein. In some examples, the genetically engineered Vibrio sp. bacteriadisclosed herein are naturally competent. In some examples, thegenetically engineered Vibrio sp. bacteria are competent cells generatedby any of the methods disclosed herein. In some examples, the competentgenetically engineered V. natriegens bacteria are generated by theprocess of: (a) growing genetically modified V. natriegens bacterialcells in a growth-conducive medium; (b) rendering said V. natriegensbacterial cells competent; and (c) freezing the cells.

In some aspects, the present disclosure provides an expression systemwhich comprises a vector comprising the Vibrio sp. chromosomalreplication machinery. In some examples, the replication machinerycomprises SEQ ID NO: 1. In some examples, the vector further comprises aheterologous nucleic acid of interest. In some examples, the vectorfurther comprises an inducible promoter operably linked to a nucleicacid of interest. In some examples, the vector is capable of replicationin E. coli or S. cerevisiae.

In some aspects, the present disclosure provides host cells comprising avector comprising Vibrio sp. chromosomal replication machinery. In someexamples, the host cells are naturally competent. In some examples, thehost cells are competent cells generated by any of the herein disclosedmethods. In some examples, the vector is introduced into the host cellby conjugation, chemical competence, natural competence, orelectroporation. In some examples, the replication machinery comprisesSEQ ID NO: 1. In some examples, the vector further comprises aheterologous nucleic acid of interest. In some examples, the vectorfurther comprises an inducible promoter operably linked to a nucleicacid of interest. In some examples, the host cells are any engineeredVibrio sp. bacteria disclosed herein.

In some aspects, the present disclosure provides a method of producing apolypeptide comprising: a) culturing cells comprising a vectorcomprising a Vibrio sp. chromosomal replication machinery and a nucleicacid encoding the polypeptide under conditions effective for theproduction of the polypeptide; and b) harvesting the polypeptide. Insome examples, the replication machinery comprises SEQ ID NO: 1. In someexamples, the vector further comprises an inducible promoter operablylinked to a nucleic acid encoding the polypeptide. In some examples, thevector is capable of replication in E. coli or S. cerevisiae. In someexamples, the cultured cells are Vibrio sp. bacterial cells. In someexamples, the Vibrio sp. cells are any of those disclosed herein. Insome examples the cultured cells or Vibrio sp. cells are naturallycompetent. In some examples, the cultured cells or Vibrio sp. cells arecompetent cells generated by any of the methods disclosed herein. Insome examples, the vector is introduced into the cultured cells orVibrio sp. cells by conjugation, chemical competence, naturalcompetence, or electroporation.

In some aspects, the present disclosure provides a method of producing apolypeptide comprising: a) contacting Vibrio sp. bacteria with a vectorcomprising a nucleic acid encoding the polypeptide and an induciblepromoter, such that the vector is introduced into the bacteria; b)growing the bacteria under conditions effective for production of thepolypeptide; and c) harvesting the polypeptide. In some examples, thevector comprises Vibrio sp. chromosomal replication machinery. In someexamples, the replication machinery comprises SEQ ID NO: 1. In someexamples, the vector is capable of replication in E. coli or S.cerevisiae. In some examples, the Vibrio sp. cells are any of thosedisclosed herein. In some examples, the Vibrio sp. bacteria arenaturally competent. In some examples the Vibrio sp. bacteria arecompetent cells generated by any of the methods disclosed herein. Insome examples, the vector is introduced into the Vibrio sp. bacteria byconjugation, chemical competence, natural competence, orelectroporation.

In some aspects, the present disclosure provides a method for cloning anucleic acid comprising: a) introducing a heterologous nucleic acid intoVibrio sp. bacteria to create a transformed bacteria; b) culturing thecells under conditions for growth of the cells; c) isolation of a singletransformed bacterial colony; d) growth of the bacterial colony; and e)extraction of nucleic acid. In some examples, the Vibrio sp. bacteriaare those of any of those disclosed herein. In some examples the Vibriosp. bacteria are naturally competent. In some examples, the Vibrio sp.bacteria are competent cells generated by any of the methods disclosedherein. In some examples, the introduction of the nucleic acid isperformed by conjugation, chemical competence, natural competence, orelectroporation. In some examples, the heterologous nucleic acid is avector. In some examples, the vector comprises Vibrio sp. chromosomalreplication machinery. In some examples, the replication machinerycomprises SEQ ID NO: 1. In some examples, the vector is capable ofreplication in E. coli or S. cerevisiae.

In some aspects, the present disclosure provides a kit for cloning DNAcomprising: a) a vector comprising the Vibrio sp. chromosomalreplication machinery; b) host cells compatible with the vector; c)buffer compatible with the host cells; and d) instructions for cloningthe DNA. In some examples, the vector further comprises an induciblepromoter. In some examples, the replication machinery comprises SEQ IDNO: 1. In some examples, the host cells are Vibrio sp. bacteria. In someexamples the Vibrio sp. bacteria are any of those disclosed herein. Insome examples, the host cells are E. coli or S. cerevisiae.

In some aspects, the present disclosure provides a kit comprisingcompetent Vibrio sp. bacterial cells. In some examples the Vibrio sp.bacterial cells are any of those disclosed herein. In some examples, thekit further comprises a compatible cloning vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the use of V. natriegens as a host for inducibleprotein expression. Six plasmids were designed for inducible proteinexpression of GFP: FIG. 1A. pBR322-trc-GFP; FIG. 1B. p15a-trc-GFP; FIG.1C. pBR322-araBAD-GFP; FIG. 1D. p15a-araBAD-GFP; FIG. 1E.pBR322-cI857ts-GFP and FIG. 1F. p15a-cI857ts-GFP.

FIG. 2 shows GFP fluorescence normalized to OD600 for induced andnon-induced cultures harboring each of the six expression plasmids.

FIG. 3 shows the presence of GFP from cultures of V. natriegensharboring pBR322-trc-GFP. Lane 1: SeeBlue Plus2 Protein Standard; Lane2: 1 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture;Lane 3: 10 μL V. natriegens pBR322-trc-GFP lysate from IPTG-inducedculture; Lane 4: 1 μL V. natriegens pBR322-trc-GFP lysate fromnon-induced culture and Lane 5: 10 μL V. natriegens pBR322-trc-GFPlysate from non-induced culture. Arrows indicate GFP.

FIG. 4 shows a plasmid comprising the sequence from V. natriegens chrII,the R6Kγ origin of replication and the tetA/tetR resistance genes alongwith the RP4 oriT region of plasmid pJB3Tc20.

FIG. 5 shows plasmid pVnoriCII-Mgen25-49 run on an agarose gel.

FIG. 6 shows the vector map and sequence of pVnatCII-YACTRP-copycontrol.

FIG. 7 shows a region of V. natriegens strain CCUG16374 chromosome Icontaining a 28 kb genetic island demarcated by the black arrow. Genesare depicted with grey arrows. The enzymes involved in the putativerestriction-modification system are demarcated by striped arrows.

FIGS. 8A-8B show V. natriegens cultures comprising T7 RNA polymeraseoperably linked to the indicated inducible promoter and a GFP cassetteoperably linked to a T7 promoter in the indicated media. FIG. 8A) Whitelight image of cultures. Two left most cultures are wild type strainsnot expressing GFP, while the other four cultures have a distinctyellow/green color, indicating expression of GFP. FIG. 8B) Blue lighttransilluminator image displaying the positive expression of GFP in theright four cultures, while the two wild type cultures on the left arenot expressing GFP and therefore lack the trademark green fluorescentcolor.

FIG. 9 shows a stained gel of bacterial lysates from wild type (two leftcultures) and the indicated four strains expressing inducible GFP. Thedark band demarcated with the asterisk is the GFP protein, which is onlypresent in the engineered strains (BHI lad, LB lad, BHI araBAD, LBaraBD), and not wild type (wt) cultures.

FIG. 10 is an illustration of the levansucrase expression vector used inconjunction with a V. natriegens strain harboring an inducible T7 RNApolymerase gene.

FIG. 11 shows a stained gel with various volumes of clarified growthmedia following a 5-hour fermentation in a minimal media with 1 mM IPTGof either V. natriegens T7 expression strain by itself (negativecontrol) or the same organism but with the levansucrase expressionplasmid, demonstrating IPTG-induced enzyme secretion. The plasmid isshown in FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to the generation of a geneticallyengineered Vibrio sp. bacteria. Specifically, the disclosure relates tothe use of the genetically engineered bacteria for the construction,maintenance, manipulation, and/or propagation of DNA constructs; proteinexpression; metabolic engineering; expression of cellular extracts forcell-free biology; and for synthetic biology applications. Thedisclosure also relates to the use of the replication machinery ofVibrio sp. on a cloning vector for replication of recombinant DNAconstructs.

Herein is described the use of the organism Vibrio natriegens as a novelhost for biotechnological applications, particularly as a host for theconstruction, maintenance, manipulation, and/or propagation ofrecombinant DNA constructs (including synthetic or semi-synthetic DNAconstructs); for protein expression; for metabolic engineering; for thepreparation of cellular extracts for cell-free biology (e.g., cell-freeprotein synthesis, in vitro enzymatic catalysis, DNA replication, andRNA transcription); and as a chassis for synthetic biology applications.Furthermore, the replication machinery of the smaller, second chromosomeof V. natriegens can be used as a novel cloning vector for replicationof recombinant DNA constructs including complete or partial exogenouschromosomes (synthetic or natural) in either V. natriegens or adifferent host such as E. coli, as well as using bacterial conjugationas a means to deliver this vector from another host into V natriegens orfrom V. natriegens to the final host organism. Applications related tomolecular biology, synthetic biology, and metabolic engineering will beaccelerated using the V. natriegens host due to its rapid growth rateand nutritional versatility.

Before the present compositions and methods are described, it is to beunderstood that this disclosure is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyin the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the disclosure, the preferred methods andmaterials are now described. The definitions set forth below are forunderstanding of the disclosure but shall in no way be considered tosupplant the understanding of the terms held by those of ordinary skillin the art.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

As used herein, “about” means either: within plus or minus 10% of theprovided value, or a value rounded to the nearest significant figure, inall cases inclusive of the provided value. Where ranges are provided,they are inclusive of the boundary values.

As used herein, “amino acid” refers to naturally-occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally-occurring amino acids.Naturally-occurring amino acids are those encoded by the genetic code,including D/L optical isomers, as well as those amino acids that arelater modified, e.g., hydroxyproline, y-carboxyglutamate, andO-phosphoserine. Amino acid analogs refer to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,a carbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally-occurring amino acid. Amino acidmimetics, as used herein, refer to chemical compounds that have astructure that is different from the general chemical structure of anamino acid, but that functions in a manner similar to anaturally-occurring amino acid.

A “nucleotide” is the basic unit of a nucleic acid molecule andtypically includes a base such as adenine, guanine, cytosine, thymine,or uracil linked to a pentose sugar such as ribose or deoxyribose thatis in turn linked to a phosphate group. Nucleotides can also includealternative or non-naturally occurring bases or sugars that do not occurin naturally-occurring DNA or RNA. In peptide nucleic acids one or moresugars may be substituted by amino acids, and in some nucleic acidanalogs at least a portion of the phosphates may be replaced by hydroxylgroups. Although nucleotides are often used to denote the length of asingle-stranded nucleic acid molecule, and “base pairs” (i.e., basepaired nucleotides) are often used to denote the length ofdouble-stranded nucleic acid molecules, in the present application,“nucleotides” or “nt” may be used interchangeably with “base pairs” or“bp”, and the use of one term or the other does not meant restrict thetype of nucleic acid molecule being described to being eithersingle-stranded or double-stranded. The use of kilobases (kb) ormegabases (Mb) as units of length also applies equally tosingle-stranded and double-stranded nucleic acid molecules.

A “nucleic acid construct”, “DNA construct” or simply “construct” is anucleic acid molecule produced by recombinant means that includes atleast two juxtaposed or operably linked nucleic acid sequences that arenot juxtaposed or operably linked to one another in nature.

A “detectable marker” is a gene or the polypeptide encoded by the genethat confers some detectable phenotype on a cell that expresses thegene. Detection can be colorometric (for example, the blue color byexpression of beta galactosidase or beta-glucuronidase in the presenceof a colorometric substrate) or by detection of luminescence orfluorescence. A detectable marker generally encodes a detectablepolypeptide, for example, a green fluorescent protein or a signalproducing enzyme such as luciferase, which, when contacted with anappropriate agent (a particular, wavelength of light or luciferin,respectively) generates a signal that can be detected by eye or usingappropriate instrumentation (Giacomin, Plant Sci. 116:59-72, 1996;Scikantha, J. Bacteriol. 178:121, 1996; Gerdes, FEBS Lett. 389:44-47,1996; see, also, Jefferson, EMBO J. 6:3901-3907, 1997).

The term or “selectable marker” or “selection marker” refers to a gene(or the encoded polypeptide) that confers a phenotype that allows theorganism expressing the gene to survive under selective conditions. Forexample, a selectable marker generally is a molecule that, when presentor expressed in a cell, provides a selective advantage (or, if anegative selectable marker, disadvantage) to the cell containing themarker, for example, the ability to grow in the presence of an agentthat otherwise would kill the cell, or the ability to grow in theabsence of a particular nutrient. Selectable markers include, but arenot limited to, an antibiotic resistance gene, a gene encoding apolypeptide conferring resistance to a toxin, an auxotrophic marker, anda combination thereof. An antibiotic resistance gene confers resistanceto antibiotics including, but is not limited to, bleomycin,carbenicillin, chloramphenicol, gentamycin, glyphosate, hygromycin,kanamycin, neomycin, nourseothricin, phleomycin, puromycin,spectinomycin, streptomycin, and tetracycline.

A “cDNA” is a DNA molecule that comprises at least a portion thenucleotide sequence of an mRNA molecule, with the exception that the DNAmolecule substitutes the nucleobase thymine, or T, in place of uridine,or U, occurring in the mRNA sequence. A cDNA can be single-stranded ordouble-stranded, and can be the complement of the mRNA sequence. Inpreferred embodiments, a cDNA does not include one or more intronsequences that occur in the naturally-occurring gene (in the genome ofan organism) that the cDNA corresponds to. For example, a cDNA can havesequences from upstream (5′) of an intron of a naturally-occurring genejuxtaposed to sequences downstream (3′) of the intron of thenaturally-occurring gene, where the upstream and downstream sequencesare not juxtaposed in a DNA molecule (i.e., the naturally occurringgene) in nature. A cDNA can be produced by reverse transcription of mRNAmolecules by a polymerase (e.g., a reverse transcriptase), or can besynthesized, for example, by chemical synthesis and/or by using one ormore restriction enzymes, one or more ligases, one or more polymerases(including, but not limited to, high temperature tolerant polymerasesthat can be used in polymerase chain reactions (PCRs)), one or morerecombinases, e.g., based on knowledge of the cDNA sequence, where theknowledge of the cDNA sequence can optionally be based on theidentification of coding regions from genome sequences and/or thesequences of one or more cDNAs.

A “coding sequence” or “coding region”, as used herein in reference toan mRNA or DNA molecule, refers to the portion of the mRNA or DNAmolecule that codes for a polypeptide. It typically consists of thenucleotide residues of the molecule which are matched with an anticodonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding sequence may includenucleotide residues corresponding to amino acid residues which are notpresent in the mature protein encoded by the mRNA molecule (e.g., aminoacid residues in a protein export signal sequence).

“Compatible” or “compatible with”, when referring to a vector orexpression system in reference to a host, refers to the vector orexpression system comprising the elements required for stablereplication within the specified host cells. Optionally, “compatible” or“compatible with” can also refer to the vector or expression systemhaving the elements and/or machinery required for one or more of thefollowing functions: transformation, propagation, maintenance,selection, and recovery from the specified host organism or system. Suchelements or machinery can comprise, but are not limited to, origins ofreplication, replication machinery, origins of transfer, transfermachinery, selectable marker, copy number induction elements, induciblepromotor, and any other elements required for transformation,conjugation, propagation, maintenance, selection, or recovery, or anyother standard scientific element or aspect.

“Competent”, ‘competence” or “competency” refers to the ability of acell to take up extracellular DNA. Competence may be “naturalcompetence”, which is a genetically specified ability of bacteria whichoccurs under natural conditions as well as in the laboratory. Competencemay alternatively be artificial or induced, which arises when cells inlaboratory cultures are treated to make them transiently permeable toDNA.

“Derived from” refers to the source of a nucleotide or amino acidsequence, and typically means the sequence of the nucleic acid molecule,protein, or peptide is based on that of the referenced nucleic acidmolecule, protein, or peptide. The nucleic acid molecule, protein, orpeptide is either a variant having at least 60% identity or homology(and, in various examples, at least 65%, at least 70%, at least 75%, atleast 80%, or at least 85% identity or homology) to the referencednucleic acid molecule, protein, or peptide, and/or is a truncated orinternally deleted variant of the referenced nucleic acid molecule,protein, or peptide. For example, a protein or peptide may beC-terminally or N-terminally truncated or internally deleted withrespect to the protein or peptide it is derived from and may have aC-terminal, N-terminal, or internal deletion of any number of aminoacids, for example, at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 amino acids. A nucleic acid molecule may be 5′ or 3′truncated or internally deleted with respect to the nucleic acidmolecule it is derived from and may have a 5′, 3′, or internal deletionof any number of nucleotides, for example, at least 1, 2, 3, 4, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides.

As used herein, the terms “percent identity” or “homology” with respectto nucleic acid or polypeptide sequences are defined as the percentageof nucleotide or amino acid residues in the candidate sequence that areidentical with the known polypeptides, after aligning the sequences formaximum percent identity and introducing gaps, if necessary, to achievethe maximum percent homology. N-terminal or C-terminal insertion ordeletions shall not be construed as affecting homology, and internaldeletions and/or insertions into the polypeptide sequence of less thanabout 30, less than about 20, or less than about 10 amino acid residuesshall not be construed as affecting homology. Homology or identity atthe nucleotide or amino acid sequence level can be determined by BLAST(Basic Local Alignment Search Tool) analysis using the algorithmemployed by the programs blastp, blastn, blastx, tblastn, and tblastx(Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990),Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored forsequence similarity searching. The approach used by the BLAST program isto first consider similar segments, with and without gaps, between aquery sequence and a database sequence, then to evaluate the statisticalsignificance of all matches that are identified, and finally tosummarize only those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul (1994), Nature Genetics 6, 119-129.The search parameters for histogram, descriptions, alignments, expect(i.e., the statistical significance threshold for reporting matchesagainst database sequences), cutoff, matrix, and filter (low complexity)can be at the default settings. The default scoring matrix used byblastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff(1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended forquery sequences over 85 in length (nucleotide bases or amino acids).

For blastn, designed for comparing nucleotide sequences, the scoringmatrix is set by the ratios of M (i.e., the reward score for a pair ofmatching residues) to N (i.e., the penalty score for mismatchingresidues), wherein the default values for M and N can be +5 and −4,respectively. Four blastn parameters can be adjusted as follows: Q=10(gap creation penalty); R=10 (gap extension penalty); wink=1 (generatesword hits at every winkth position along the query); and gapw=16 (setsthe window width within which gapped alignments are generated). Theequivalent Blastp parameter settings for comparison of amino acidsequences can be: Q=9; R=2; wink=1; and gapw=32. A Bestfit comparisonbetween sequences, available in the GCG package version 10.0, can useDNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extensionpenalty), and the equivalent settings in protein comparisons can beGAP=8 and LEN=2. Thus, when referring to the polypeptide or nucleic acidsequences of the present disclosure, included are sequence identities ofat least 40%, at least 45%, at least 50%, at least 55%, of at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%,for example at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orabout 100% sequence identity with the full-length polypeptide or nucleicacid sequence, or to fragments thereof comprising a consecutive sequenceof at least 100, at least 125, at least 150 or more amino acid residuesof the entire protein; variants of such sequences, e.g., wherein atleast one amino acid residue has been inserted N- and/or C-terminal to,and/or within, the disclosed sequence(s) which contain(s) the insertionand substitution. Contemplated variants can additionally or alternatelyinclude those containing predetermined mutations by, e.g., homologousrecombination or site-directed or PCR mutagenesis, and the correspondingpolypeptides or nucleic acids of other species, including, but notlimited to, those described herein, the alleles or other naturallyoccurring variants of the family of polypeptides or nucleic acids whichcontain an insertion and substitution; and/or derivatives wherein thepolypeptide has been covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid which contains the insertion andsubstitution (for example, a detectable moiety such as an enzyme).

As used herein, the phrase “conservative amino acid substitution” or“conservative mutation” refers to the replacement of one amino acid byanother amino acid with a common property. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz (1979) Principles of ProteinStructure, Springer-Verlag). According to such analyses, groups of aminoacids can be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on the overall protein structure (Schulz (1979)Principles of Protein Structure, Springer-Verlag). Examples of aminoacid groups defined in this manner can include: a “charged/polar group”including Glu, Asp, Asn, Gln, Lys, Arg, and His; an “aromatic or cyclicgroup” including Pro, Phe, Tyr, and Trp; and an “aliphatic group”including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within eachgroup, subgroups can also be identified. For example, the group ofcharged/polar amino acids can be sub-divided into sub-groups including:the “positively-charged sub-group” comprising Lys, Arg and His; the“negatively-charged sub-group” comprising Glu and Asp; and the “polarsub-group” comprising Asn and Gln. In some examples, the aromatic orcyclic group can be sub-divided into sub-groups including: the “nitrogenring sub-group” comprising Pro, His, and Trp; and the “phenyl sub-group”comprising Phe and Tyr. In another further example, the aliphatic groupcan be sub-divided into sub-groups including: the “large aliphaticnon-polar sub-group” comprising Val, Leu, and Ile; the “aliphaticslightly-polar sub-group” comprising Met, Ser, Thr, and Cys; and the“small-residue sub-group” comprising Gly and Ala. Examples ofconservative mutations include amino acid substitutions of amino acidswithin the sub-groups above, such as, but not limited to: Lys for Arg orvice versa, such that a positive charge can be maintained; Glu for Aspor vice versa, such that a negative charge can be maintained; Ser forThr or vice versa, such that a free —OH can be maintained; and Gln forAsn or vice versa, such that a free —NH2 can be maintained. A“conservative variant” is a polypeptide that includes one or more aminoacids that have been substituted to replace one or more amino acids ofthe reference polypeptide (for example, a polypeptide whose sequence isdisclosed in a publication or sequence database, or whose sequence hasbeen determined by nucleic acid sequencing) with an amino acid havingcommon properties, e.g., belonging to the same amino acid group orsub-group as delineated above.

The term “essential”, when referring to a gene or element, means a geneor element of an organism that are thought to be critical for thesurvival of the organism. For example, essential genes can encodeproteins or RNAs that maintain central metabolism, replicate DNA,transcribe and translate genes into proteins, maintain a basic cellularstructure, and mediate transport processes into and out of the cell.“Conditionally essential” genes or elements are those that are requiredor essential under certain circumstances, for instance, a gene requiredto digest starch is only essential if starch is the only energy sourceavailable to the organism. “Non-essential” genes or elements oftenconvey a selective advantaged or increased fitness for the organism incertain circumstances, but are not absolutely required for life.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to anucleotide sequence of interest, which can optionally be operably linkedto termination signals and/or other regulatory elements. An expressioncassette may also comprise sequences that enable, mediate, or enhancetranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a non-translated RNA, in thesense or antisense direction. An expression cassette may be assembledentirely extracellularly (e.g., by recombinant cloning techniques).However, an expression cassette may also be assembled using in partendogenous components. For example, an expression cassette may beobtained by placing (or inserting) a promoter sequence upstream of anendogenous sequence, which thereby becomes functionally linked andcontrolled by said promoter sequences. The expression of the nucleotidesequence in the expression cassette may be under the control of aconstitutive promoter or of an inducible promoter which initiatestranscription only when the host cell is exposed to some particularexternal stimulus.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Examples of expression vectors known in the artinclude cosmids, plasmids and viruses (e.g., retroviruses, lentiviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Exogenous nucleic acid molecule” or “exogenous gene” refers to anucleic acid molecule or gene that has been introduced (“transformed”)into a cell. A transformed cell may be referred to as a recombinant cellor an engineered cell. A nucleic acid molecule is also exogenous if itis present in a descendent cell and received from an ultimate parentcell where that nucleic acid molecule was exogenous nucleic acid. Theexogenous gene may be from a different species (thus also“heterologous”), or from the same species (thus “homologous”), relativeto the cell being transformed.

The term “heterologous” when used in reference to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide,gene, nucleic acid, polypeptide, or enzyme that is from a source orderived from a source other than the host organism species. Heterologousmolecules are therefore always also exogenous, but exogenous moleculesare not necessarily always heterologous. In contrast a “homologous”polynucleotide, gene, nucleic acid, polypeptide, or enzyme is usedherein to denote a polynucleotide, gene, nucleic acid, polypeptide, orenzyme that is derived from the host organism species. When referring toa gene regulatory sequence or to an auxiliary nucleic acid sequence usedfor maintaining or manipulating a gene sequence (e.g., a promoter, a 5′untranslated region, 3′ untranslated region, poly A addition sequence,intron sequence, splice site, ribosome binding site, internal ribosomeentry sequence, genome homology region, recombination site, etc.),“heterologous” means that the regulatory sequence or auxiliary sequenceis not naturally associated with the gene with which the regulatory orauxiliary nucleic acid sequence is juxtaposed in a construct, genome,chromosome, or episome. Thus, a promoter operably linked to a gene towhich it is not operably linked to in its natural state (i.e., in thegenome of a non-genetically engineered organism) is referred to hereinas a “heterologous promoter,” even though the promoter may be derivedfrom the same species (or, in some cases, the same organism) as the geneto which it is linked.

A “recombinant” or “engineered” or “genetically engineered” nucleic acidmolecule, polypeptide, organism, or combination thereof is a nucleicacid molecule, polypeptide, organism, or combination thereof that hasbeen altered through human manipulation. As non-limiting examples, arecombinant nucleic acid molecule includes any nucleic acid moleculethat: 1) has been partially or fully synthesized or modified in vitro,for example, using chemical or enzymatic techniques (e.g., by use ofchemical nucleic acid synthesis, or by use of enzymes for thereplication, polymerization, digestion (exonucleolytic orendonucleolytic), ligation, reverse transcription, transcription, basemodification (including, e.g., methylation), integration orrecombination (including homologous and site-specific recombination) ofnucleic acid molecules); 2) includes conjoined nucleotide sequences thatare not conjoined in nature, 3) has been engineered using molecularcloning techniques such that it lacks one or more nucleotides withrespect to the naturally occurring nucleic acid molecule sequence,and/or 4) has been manipulated using molecular cloning techniques suchthat it has one or more sequence changes or rearrangements with respectto the naturally occurring nucleic acid sequence. As non-limitingexamples, a cDNA is a recombinant DNA molecule, as is any nucleic acidmolecule that has been generated by in vitro polymerase reaction(s), orto which linkers have been attached, or that has been integrated into avector, such as a cloning vector or expression vector.

“Minimize/d” or “minimization” as used herein when referring to agenome, chromosome, or nucleic acid sequence, refers to removingnon-essential nucleic acid sequences and/or rearranging the order ofnucleic acid sequences which results in a smaller nucleic acid moleculethan what was originally started with.

An “oligonucleotide”, as used herein, is a nucleic acid molecule 200 orfewer nucleotides in length. An oligonucleotide can be RNA, DNA, or acombination of DNA and RNA, a nucleic acid derivative, or a syntheticnucleic acid, for example, an oligonucleotide can be a peptide nucleicacid or a locked nucleic acid, and can be single-stranded,double-stranded, or partially single-stranded and partiallydouble-stranded. An oligonucleotide can be, for example, between about 4and about 200 nucleotides in length, between about 6 and about 200nucleotides in length, between about 10 and about 200 nucleotides inlength, between about 15 and about 200 nucleotides in length, betweenabout 17 and about 200 nucleotides in length, between about 20 and about200 nucleotides in length, or between about 40 and about 200 nucleotidesin length. In additional examples, an oligonucleotide can be betweenabout 15 and about 180 nucleotides in length, between about 15 and about160 nucleotides in length, between about 15 and about 140 nucleotides inlength, between about 15 and about 120 nucleotides in length, betweenabout 17 and about 100 nucleotides in length, between about 17 and about80 nucleotides in length, or between about 17 and about 70 nucleotidesin length, for example between about 20 and about 65 nucleotides inlength, or between about 40 and about 80 nucleotides in length.

When used in reference to a polynucleotide, a gene, a nucleic acid, apolypeptide, or an enzyme, the term “heterologous” refers to apolynucleotide, gene, a nucleic acid, polypeptide, or an enzyme notderived from the host species, e.g., from a different species withrespect to the host cell. When referring to nucleic acid sequencesoperably linked or otherwise joined to one another (“juxtaposed”) in anucleic acid construct or molecule, “heterologous sequences”, as usedherein, are those that are not operably linked or are not in proximityor contiguous to each other in nature. Similarly, when referring to agene regulatory sequence or to an auxiliary nucleic acid sequence usedfor maintaining or manipulating a gene sequence (e.g., a 5′un-translated region, 3′ un-translated region, Kozak sequence, poly Aaddition sequence, intron sequence, splice site, ribosome binding site,internal ribosome entry sequence, genome homology region, recombinationsite, e.g.), “heterologous” means that the regulatory sequence orauxiliary sequence is from a different source (e.g., different gene,whether from the same or different species as the host organisms) thanthe gene with which the regulatory or auxiliary nucleic acid sequence isjuxtaposed or operably linked in a construct, genome, chromosome, orepisome.

The terms “nucleic acid molecule” and “polynucleotide molecule” are usedinterchangeably herein, and refer to both DNA and RNA molecule,including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containingnucleic acid analogs. Polynucleotides can have any three-dimensionalstructure. Polynucleotides can be natural-occurring or synthetic origin.A nucleic acid molecule can be double-stranded or single-stranded.Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), siRNA, micro-RNA, ribozymes, tracr RNAs, crRNAs, chimeric guideRNAs, cDNA, recombinant polynucleotides, branched polynucleotides,nucleic acid probes and nucleic acid primers. A polynucleotide maycontain unconventional or modified nucleotides.

As used herein, “operably linked” is intended to mean a functionallinkage between two or more sequences such that activity at or on onesequence affects activity at or on the other sequence(s). For example,an operable linkage between a polynucleotide of interest and aregulatory sequence (e.g., a promoter) is a functional link that allowsfor expression of the polynucleotide of interest. In this sense, theterm “operably linked” refers to the positioning of a regulatory regionand a coding sequence to be transcribed so that the regulatory region iseffective for regulating transcription or translation of the codingsequence of interest. For example, to operably link a coding sequenceand a regulatory region, the translation initiation site of thetranslational reading frame of the coding sequence is typicallypositioned between one and about fifty nucleotides downstream of theregulatory region. A regulatory region can, however, be positioned asmuch as about 5,000 nucleotides upstream of the translation initiationsite, or about 2,000 nucleotides upstream of the transcription startsite. Operably linked elements may be contiguous or non-contiguous. Whenused to refer to the joining of two protein coding regions, by “operablylinked” is intended that the coding regions are in the same readingframe. When used to refer to the effect of an enhancer, “operablylinked” indicated that the enhancer increases the expression of aparticular polypeptide or polynucleotides of interest. “Juxtaposed with”in the context of nucleic acid sequences, means the referenced sequencesare part of the same continuous nucleic acid molecule.

The terms “polynucleotide sequence” and “nucleic acid sequence” as usedherein interchangeably refer to a sequence of a polynucleotide molecule,and can refer, for example, to DNA or RNA sequences. The nomenclaturefor nucleotide bases as set forth in 37 CFR § 1.822 is used herein.

The term “promoter” refers to a nucleic acid sequence capable of bindingRNA polymerase in a cell and initiating transcription of a downstream(3′ direction) coding sequence. A promoter includes the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. A promoter can include a transcriptioninitiation site as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promotersoften, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryoticpromoters may contain −10 and −35 prokaryotic promoter consensussequences. A large number of promoters, including constitutive,inducible and repressible promoters, from a variety of different sourcesare well known in the art. Representative sources include for example,algal, viral, mammalian, insect, plant, yeast, and bacterial cell types,and suitable promoters from these sources are readily available, or canbe made synthetically, based on sequences publicly available on line or,for example, from depositories such as the ATCC as well as othercommercial or individual sources. Promoters can be unidirectional(initiate transcription in one direction) or bi-directional (initiatetranscription in either direction). A promoter may be a constitutivepromoter, a repressible promoter, or an inducible promoter. Suchpromoters need not be of naturally-occurring sequences. In addition, itwill be understood that such promoters need not be derived from thetarget host cell or host organism.

“Polypeptide” and “protein” are used interchangeably herein and refer toa compound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics, regardless of post-translational modification,e.g., phosphorylation or glycosylation. The subunits may be linked bypeptide bonds or other bonds such as, for example, ester or ether bonds.Full-length polypeptides, truncated polypeptides, point mutants,insertion mutants, splice variants, chimeric proteins, and fragmentsthereof are encompassed by this definition. In various embodiments thepolypeptides can have at least 10 amino acids or at least 25, or atleast 50 or at least 75 or at least 100 or at least 125 or at least 150or at least 175 or at least 200 amino acids.

As used herein the term “biomolecule” means any molecule that is presentin living organisms or that is produced by living organisms, includinglarge macromolecules such as proteins, polysaccharides, lipids, andnucleic acids, as well as small molecules such as primary metabolites,secondary metabolites, and natural products. A more general name forthis class of material is biological materials.

As used herein “progeny” means a descendant, offspring, or derivative ofan organism. For example, daughter cells from a transgenic alga areprogeny of the transgenic alga. Because certain modifications may occurin succeeding generations due to mutations or environmental influences,such progeny, descendant, or derivatives may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The terms “recombinant” or “engineered” as used herein in reference to anucleic acid molecule, refer to a nucleic acid molecule that has beenaltered through human intervention. As non-limiting examples, a cDNA isa recombinant DNA molecule, as is any nucleic acid molecule that hasbeen generated by in vitro polymerase reaction(s), or to which linkershave been attached, or that has been integrated into a vector, such as acloning vector or expression vector. As non-limiting examples, arecombinant nucleic acid molecule: 1) has been synthesized or modifiedin vitro, for example, using chemical or enzymatic techniques (forexample, by use of chemical nucleic acid synthesis, or by use of enzymesfor the replication, polymerization, exonucleolytic digestion,endonucleolytic digestion, ligation, reverse transcription,transcription, base modification (including, e.g., methylation), orrecombination (including homologous and site-specific recombination)) ofnucleic acid molecules; 2) includes conjoined nucleotide sequences thatare not conjoined in nature; 3) has been engineered using molecularcloning techniques such that it lacks one or more nucleotides withrespect to the naturally occurring nucleic acid molecule sequence;and/or 4) has been manipulated using molecular cloning techniques suchthat it has one or more sequence changes or rearrangements with respectto the naturally occurring nucleic acid sequence. A “recombinantprotein” is a protein produced by genetic engineering, for example, byexpression of a genetically engineered nucleic acid molecule in a cell.

The term “regulatory region”, “regulatory sequence”, “regulatoryelement”, or “regulatory element sequence”, as used in the presentdisclosure, refer to a nucleotide sequence that influences transcriptionor translation initiation or rate, and stability and/or mobility of atranscription or translation product. Such regulatory regions need notbe of naturally-occurring sequences. Regulatory sequences include butare not limited to promoter sequences, enhancer sequences, responseelements, protein recognition sites, inducible elements, protein bindingsequences, 5′ and 3′ un-translated regions (UTRs), transcriptional startsites, termination sequences, polyadenylation sequences, introns, andcombinations thereof. A regulatory region typically comprises at least acore (basal) promoter. A regulatory region also may include at least onecontrol element, such as an enhancer sequence, an upstream element or anupstream activation region (UAR).

As used herein, the terms “chromosomal replication machinery” or“replication machinery” mean that part of an organism's chromosome whichsupports replication within the organism or in a different organism. Insome aspects of the present disclosure, replication machinery refers a5-6 kb or 5-5.5 kb or about 5.5 kb sequence from chromosome II of V.natriegens which is capable of supporting replication in an organism. Incertain aspects, the replication machinery from V. natriegens cansupport replication in V. natriegens and E. coli. In other aspects ofthe present disclosure, the replication machinery has at least about60%, at least about 70%, at least about 80%, at least about 90%, or atleast about 95%, or at least about 97%, or at least about 98%, or atleast about 99% sequence identity or homology to the sequence of SEQ IDNO: 1 or a variant thereof. SEQ ID NO: 1 comprises an origin ofreplication operable in Vibrio sp. and in E. coli.

As used herein, “transgenic organism” refers to an organism whichcomprises a heterologous polynucleotide. When applied to organisms, theterms “transgenic” or “recombinant” or “engineered” or “geneticallyengineered,” used interchangeably herein, refer to organisms that havebeen manipulated by introduction into the organism of an exogenous orrecombinant nucleic acid sequence. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations, although it canalso be present on an episome, and may be present on a syntheticchromosome of the transgenic organism. The non-native polynucleotide maybe integrated into the genome alone or as part of a recombinantexpression cassette. In additional examples, a transgenic microorganismcan include an introduced exogenous regulatory sequence operably linkedto an endogenous gene of the transgenic microorganism. Non-limitingexamples of such manipulations include gene knockouts, targetedmutations and gene replacement, promoter replacement, deletion, orinsertion, as well as introduction of transgenes into the organism.Recombinant or genetically engineered organisms can also be organismsinto which constructs for gene “knock down” have been introduced. Suchconstructs include, but are not limited to, RNAi, microRNA, shRNA,antisense, and ribozyme constructs. Also included are organisms whosegenomes have been altered by the activity of meganucleases, TALENs, zincfinger nucleases, or CRISPR nucleases. As used herein, “recombinantmicroorganism” or “recombinant host cell” includes progeny orderivatives of the recombinant microorganisms of the disclosure. Becausecertain modifications may occur in succeeding generations from eithermutation or environmental influences, such progeny or derivatives maynot, in fact, be identical to the parent cell, but are still includedwithin the scope of the term as used herein.

For nucleic acids and polypeptides, the term “variant” is used herein todenote a polypeptide, protein, or polynucleotide molecule with somedifferences, generated synthetically or naturally, in their base oramino acid sequences as compared to a reference polypeptide orpolynucleotide, respectively, such that the variant has at least 70%sequence identity with the reference polypeptide or polynucleotide. Inother embodiments the variant can have at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity with the reference polypeptide orpolynucleotide. In other embodiments the variant has a sequence identityof at least 70% or at least 80% or at least 90% or at least 95% or atleast 97% or at least 98% or at least 99% or 90-99% or 95-99% or 97-99%to a sequence of at least 5 or at least 7 or at least 10 or at least 15or at least 20 or at least 30 or at least 40 or at least 50 or at least100 or at least 200 or at least 300 or at least 400 or at least 500 orat least 600 or at least 700 or at least 800 or at least 900 or at least1000 or at least 2000 or at least 3000 at least 4000 or at least 5000consecutive nucleotides or amino acids from the reference sequence(e.g., SEQ ID NO: 1-25 or any sequence described herein). Alternatively,or in addition, a variant can have one or two or three or four or fiveor six or seven or eight or nine or ten or more insertions or deletionsin response to a reference polypeptide or polynucleotide. For example,protein variants may be N-terminally truncated or C-terminally truncatedwith respect to the reference sequence, or can have one or more internaldeletions, while nucleic acid variants may have a 5′ end and/or 3′endsequence truncation and/or can have one or more internal deletions.Further, a protein variant may have an additional sequence added to theN-terminus and/or C-terminus with respect to the reference sequence, orcan have one or more internal additional sequences, while nucleic acidvariants may have a 5′ end and/or 3′end sequence addition and/or canhave one or more internal sequence additions. A variant can have anydesired combination of substitutions, insertions, and/or deletions withrespect to a reference polypeptide or polynucleotide. Polypeptide andprotein variants can further include differences in post-translationalmodifications (such as glycosylation, methylation, phosphorylation,e.g.). When the term “variant” is used in reference to a microorganism,it typically refers to a strain microbial strain having identifyingcharacteristics of the species to which it belongs, while having atleast one nucleotide sequence variation or identifiably different traitwith respect to the parental strain, where the trait is geneticallybased (heritable).

A “vector” is any genetic element capable of serving as a vehicle ofgenetic transfer, expression, or replication for a foreignpolynucleotide in a host cell. For example, a vector may be anartificial chromosome or a plasmid, and may be capable of stableintegration into a host cell genome, or it may exist as an independentgenetic element (e.g., episome, plasmid). A vector may exist as a singlepolynucleotide or as two or more separate polynucleotides. Vectors maybe single copy vectors or multicopy vectors when present in a host cell.

Vibrio is a genus of Gram-negative, facultative anaerobic bacteriapossessing a curved-rod shape. In some embodiments, Vibrio sp. comprisesone or more of the following Vibrio species: adaptatus, aerogenes,aestivus, aestuarianus, agarivorans, albensis, alfacsensis,alginolyticus, anguillarum, areninigrae, artabrorum, atlanticus,atypicus, azureus, brasiliensis, bubulus, calviensis, campbellii, casei,chagasii, cholera, cincinnatiensis, coralliilyticus, crassostreae,cyclitrophicus, diabolicus, diazotrophicus, ezurae, fischeri, fluvialis,fortis, furnissii, gallicus, gazogenes, gigantis, halioticoli, harveyi,hepatarius, hippocampi, hispanicus, hollisae, ichthyoenteri, indicus,kanaloae, lentus, litoralis, logei, mediterranei, metschnikovii,mimicus, mytili, natriegens, navarrensis, neonates, neptunius, nereis,nigripulchritudo, ordalii, orientalis, pacinii, parahaemolyticus,pectenicida, penaeicida, pomeroyi, ponticus, proteolyticus,rotiferianus, ruber, rumoiensis, salmonicida, scophthalmi, splendidus,superstes, tapetis, tasmaniensis, tubiashii, vulnificus, wodanis, andxuii. In some embodiments, Vibrio sp. is not Vibrio cholera. In someembodiments, Vibrio sp. comprises all known species of Vibrio other thancholera. In some embodiments, Vibrio sp. comprises Vibrio natriegens. Ina preferred embodiment, Vibrio sp. is Vibrio natriegens.

Vibrio natriegens is a Gram-negative marine bacterium. It was firstisolated from salt marsh mud and is a halophile requiring about 2% NaClfor growth. It reacts well to the presence of sodium ions which appearto stimulate growth in Vibrio species, to stabilize the cell membrane,and to affect sodium-dependent transport and mobility. Under optimumconditions, and all nutrients provided, the doubling time of V.natriegens can be less than 10 minutes. Its rapid growth rate (thefastest known doubling time of any organism), its ability to thrive ininexpensive, defined media, its ability to serve as a drop-inreplacement for E. coli strains for common lab processes, its uniquegenome architecture (which can be leveraged to facilitate the cloning oflarge DNAs), and the potential to leverage natural transformation andconjugation as genetic engineering tools makes V. natriegens anattractive host. It has the potential to dramatically speed up standardworkflows, as well as to make possible projects that are too ambitiousfor the current state of the art.

Vibrio sp. has several advantages over other bacteria for many molecularbiology applications. One such advantage is the rapid growth rate ofVibrio sp. One of the most time intensive steps in modern biotechworkflows is in waiting for the host to grow to a sufficient densitybefore DNA/protein/product can be recovered or the phenotype can beassessed. As dramatic time savings have been realized in other areas ofbiotech workflows (e.g., sequencing, bioinformatic analysis,high-throughput assays, etc.), growth of the host has become asignificant bottleneck. E. coli is considered to have one of thequickest growth rates relative to other organisms used in the biotechsector, which has been one of its strengths. Because Vibrio natriegenshas a growth rate 2-3× faster than commonly used E. coli strains, it isable to effect a dramatic reduction in the time necessary for the hostto grow, and will accelerate research efforts. In certain aspects of thepresent disclosure, the growth rate of Vibrio sp. is about 10 minutes.In other aspects, the growth rate of a genetically engineered Vibrio sp.is about 5 minutes to 30 minutes. In various embodiments the Vibrio sp.organisms of the invention have a doubling time of less than 15 minutesor less than 14 minutes or less than 13 minutes or less than 12 minutesor less than 11 minutes or less than 10 minutes, or less than 9 minutes.The doubling time can be achieved by the organism in a rich media,meaning that it is rich in nitrogen and carbon. The doubling timesdescribed can be achievable in any of the media described herein (e.g.,any described in Example 1). In specific examples the doubling timesdisclosed can be achieved in an LB broth, in LB agar, in NutrientBroth+1.5% NaCl, in Brain Heart Infusion (with or without salts), BrainHeart Infusion Agar (with or without salts), SSG agar,2xYT+salts+glucose+phosphate buffer, Vegitone Infusion Broth (withoptional salts), LB+salts+glucose+phosphate buffer. Example formulationsof these media are described in Example 1. Any of the salts or othermedia components can be as described herein, for example as inExample 1. In various embodiments doubling times can be measured at theflat or log portion of the curve.

Another advantage is the size of exogenous DNA that can be harbored inVibrio sp. Large scale genetic engineering/synthetic genome constructionefforts require the assembly, manipulation, and maintenance of largepieces of recombinant DNA, tasks which are carried out in a geneticallytractable host (such as E. coli) before delivery of the engineered DNAto the final host organism. Currently, most of this work is carried outin E. coli, but as projects become more ambitious, the limitations ofthis species are becoming apparent. It has been observed that withcurrent technologies, E. coli is capable of harboring exogenous DNAconstructs of no more than 500 kb (and in some cases much less dependingon the properties of the DNA being cloned) on a bacterial artificialchromosome, which is a serious limitation for synthetic genome/largepathway construction efforts. This has necessitated the development ofnovel hosts as cloning platforms such as Saccharomyces cerevisiae andBacillus subtilis. While these hosts have the advantage of being able totake up and stably propagate large (Mb-sized) fragments of exogenousDNA, they have their own disadvantages, with Saccharomyces cerevisiaegrowing much slower than E. coli (˜3× slower), and both species beingincompatible with standard laboratory techniques and being verydifficult to recover DNA from.

An additional advantage is the compatibility of Vibrio sp. with standardlab protocols: Unlike other niche organisms, which often requirespecialized techniques/methods, Vibrio sp. is compatible with manystandard cloning vectors, growth media, workflows andcommercially-available kits developed for E. coli or recovering DNA.This compatibility with standard tools/reagents/methods lowers thebarrier to adoption by labs that are currently dependent on E. coli,allowing for drop-in replacement.

A further advantage is the nutritional versatility of Vibrio sp. Oneadditional benefit of Vibrio sp. is its extreme nutritional versatility,allowing it to grow on a range of different growth media, includinginexpensive, minimal media. Coupled with its rapid growth rate, thisfeature will allow for industrial scale production of biomolecules(e.g., therapeutic proteins, commodity chemicals, etc.) cheaper andfaster than the state of the art. As described in the Examples, V.natriegens and a genetically engineered V. natriegens are capable ofgrowing under a variety of nutrient and temperature conditions.

Members of Vibrionaceae have a unique two-chromosome genome, hereinreferred to as Chromosome I and Chromosome II. A genetically engineeredV. natriegens can be constructed with a single, large chromosome whichincorporates the essential features from the smaller chromosome into thelarge chromosome. In this genetically engineered V. natriegens, the now“free” chromosomal machinery can be leveraged as a vector for cloninglarge DNAs/pathways. The smaller second chromosome of V. natriegens canbe capable of replicating/maintaining a ˜2 Mb fragment of DNA showingthat the use of chromosome II as a cloning vector can allow for therapid and robust propagation of large exogenous DNA molecules (e.g.,synthetic or semi-synthetic chromosomes for ultimate use in otherorganisms, or novel pathways/genetic elements for use in V. natriegensitself) as well as the production of polypeptides and biomolecules.

In some embodiments, the present disclosure provides geneticallyengineered Vibrio sp. bacteria comprising one or two altered,rearranged, or minimized chromosomes. In some examples, the essentialelements from Chromosome II are alternatively located on Chromosome I.In some examples, the engineered bacteria contain a single chromosomecomprising the essential features of Chromosome I and II. In someexamples, the engineered Vibrio sp. are generated by knocking out and/orknocking in appropriate genes in order to generate the desiredengineered Vibrio sp. In some examples the knock in and/or knock outand/or sequence inversion is enabled through the enzyme activity of arecombinase, such as, for example, Cre recombinase. In some examples,the Cre recombinase activity utilizes known lox sites compatible withCre recombinase. In some examples, the knock in and/or knock out isenabled through the enzyme activity of a nuclease, such as, for example,Type II CRISPR Cas9. In some examples, the knock in and/or knock out isenabled through the use of a homologous recombination vector containingregions of sequence homology to a region in the genome where aninsertion or deletion is desired. In some examples, the homologousrecombination vector is incorporated by a single cross-over event. Insome examples, the homologous recombination vector is incorporated by adouble-cross-over event. In some examples the knock in and/or knock outevent is enables through use of an integrase, such as, for example,PhiC31 or bxb1. In some examples, the knock in and/or knock out isenabled through the use of a suicide vector. In some examples the vectoris assembled in vitro and subsequently transformed and amplified in E.coli. In some examples the vector is assembled in S. cerevisiae. In someexamples, the amplified vector is introduced into V natriegens byconjugation, electroporation, chemical competence, biolistics,transduction, or via natural competence.

In some examples, the genetically engineered Vibrio sp. comprisesaltered chromosomes or a combined single chromosome, either of which hasbeen minimized, whereby non-essential genes and nucleic acid sequenceshave been removed. Non-limiting examples of non-essential genes includeexonucleases, endonucleases, methylases, nucleases, restriction enzymes,complete restriction-modification systems, or any combination thereof.Non-essential genes or genetic elements can be identifiedbioinformatically or experimentally. Bioinformatic identification caninvolve comparing multiple wild types V. natriegens strain genomes andidentifying genes or nucleic acid sequences that are not consistentlypresent in all strains. Experimental identification of non-essentialgenes can be achieved by transposon bombardment or other insertionalmutagenesis screens that will produce multiple random integrationmutants. By sequencing the genes disrupted in these viable mutants,non-essential genes will be identified. In some examples, thenon-essential genes can be sequentially removed by homologousrecombination based techniques. In some examples, the minimization canbe achieved sequentially through known techniques, such as multiplexautomated genome engineering (MAGE) or hierarchical conjugative assembly(CAGE).

In some embodiments, the present disclosure provides geneticallyengineered Vibrio sp. bacteria comprising an altered chromosomalarrangement. In some aspects, one or more non-essential elements areremoved from Chromosome I and/or Chromosome II. In some aspects, one ormore elements from Chromosome II are alternatively located on ChromosomeI. In some aspects, one or more elements from Chromosome II arealternatively located on Chromosome I. In some aspects, the geneticallyengineered Vibrio sp. comprises a single chromosome. In some aspects,the single chromosome contains essential genomic elements from Vibriosp. Chromosome I and II. In some examples, non-limiting examples of anessential element is a gene required for a function selected from thegroup consisting of metabolism, DNA replication, transcription,translation, cellular structural maintenance, transport processes intoor out of the cell, or any combination thereof.

In some aspects, the one or two chromosomes are “minimized”, wherebynon-essential elements have been removed. In some aspects, the bacteriagrow at temperature from about 25° C. to about 42° C. In some aspects,the growth doubling time is about 5 minutes to 15 minutes. In someexamples, the minimized chromosome or single chromosome comprisesessential elements from Chromosome I and Chromosome II such that theminimized or single chromosome is capable of supporting survival andreplication of the bacteria under non-selective conditions.

In some aspects, the herein disclosed genetically engineered Vibrio sp.further comprises a heterologous nucleic acid sequence operably linkedto a heterologous promoter. In some examples the heterologous nucleicacid encodes T7 RNA polymerase. In some examples, the heterologouspromoter is an inducible promoter. The inducible promoter can be inducedby temperature, arabinose, or IPTG as non-limiting examples.

In some embodiments, the present disclosure provides a process forproducing competent Vibrio sp. cells comprising growing geneticallymodified Vibrio sp. bacterial cells in a growth-conducive medium;rendering said Vibrio sp. bacterial cells competent; and freezing thecells. In some aspects, the Vibrio sp. are any of those geneticallyengineered Vibrio sp. described herein. In some aspects, rendering thecells competent comprises growing the cells in conducive mediasupplemented with supplemental salts.

In some embodiments, the present disclosure provides a method ofproducing a biomolecule comprising a) providing a Vibrio sp. having anexogenous nucleic acid that comprises a heterologous nucleic acidsequence encoding the biomolecule. The method can, optionally, include astep of contacting the Vibrio sp. with the exogenous nucleic acid andintroducing it into the bacteria; the exogenous nucleic acid can be aplasmid, expression vector, or other vector that encodes a heterologousnucleic acid sequence; b) growing the bacteria in a growth-conducivemedium wherein the heterologous nucleic acid sequence is expressed,thereby producing the biomolecule; and optionally c) isolating thebiomolecule. In some embodiments the exogenous nucleic acid can encode asignal sequence that causes the biomolecule to be secreted from theorganism when produced. The biomolecule can therefore be expressed witha signal sequence attached. In some examples, the bacteria are any ofthe genetically engineered Vibrio sp. bacteria disclosed herein, forexample a Vibrio natriegens. In some examples, the exogenous nucleicacid comprises a nucleic acid sequence encoding Vibrio sp. replicationmachinery of SEQ ID NO: 1 or a variant thereof. In some examples, theexogenous nucleic acid further comprises an inducible promoter operablylinked to the heterologous nucleic acid encoding the biomolecule. Insome aspects, the exogenous nucleic acid comprises replication machinerycompatible with one or more organisms. In some examples the replicationmachinery is compatible with a heterologous host, such as, for example,E. coli or S. cerevisiae. Alternatively, or additionally, thereplication machinery is from V. natriegens. In some aspects, theheterologous nucleic acid is at least 1 kb or at least 10 kb, or atleast 25 kb, or at least 50 kb, or at least 75 kb, or at least 100 kb,or at least 125 kb, or at least 150 kb, or at least 175 kb, or at least200 kb, or at least 250 kb, or at least 300 kb, or at least 350 kb, orat least 400 kb, or at least 500 kb, or at least 600 kb, or at least 700kb, or at least 800 kb, or at least 900 kb or at least 1 Mb, or 2 Mb, or3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1 Mb or 50 kb-1 Mb or 100kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5 Mb or 50 kb-7 Mb, or 30kb-1 Mb or 100 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5 Mb or 30kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100 kb-7 Mb. Ina specific aspect, the heterologous nucleic acid is at least about 500kb. In some aspects, the heterologous nucleic acid also comprises aninducible promoter, an origin of replication, an origin of transfer, aselectable marker, a counter-selectable marker, a reporter gene, aregulatory element, an enzyme gene, or a combination thereof. In someaspects, the selectable marker is an antibiotic resistance gene, a geneencoding a polypeptide conferring resistance to a toxin, an auxotrophicmarker or a combination thereof. In some aspects, the antibioticresistance gene confers resistance to antibiotics which is bleomycin,carbenicillin, chloramphenicol, gentamycin, glyphosate, hygromycin,kanamycin, neomycin, nourseothricin, phleomycin, puromycin,spectinomycin, streptomycin, or tetracyclin. In some examples, thereporter gene can be a fluorescent protein or beta-galactosidase. Insome aspects, the enzyme is a recombinase, integrase, nuclease,recombineering enzymes, or polymerase. In some examples the recombinaseis Cre recombinase. In some examples, the integrase is PhiC31 or bxb1.In some examples the nuclease is a TypeII CRISPR Cas9 nuclease. In someexamples, the polymerase is a Sp6, T3, or T7 RNA polymerase. In someaspects, the inducible promoter is induced by IPTG, arabinose, ortemperature. In some examples, the Vibrio sp. bacterial cells arenaturally competent. In some examples, the Vibrio sp. cells arecompetent cells generated by any of the methods disclosed herein. Insome aspects, the heterologous nucleic acid is introduced into the cellby conjugation, chemical competence, natural competence, biolistics,transduction, or electroporation. In some aspects, the growth conducivemedia is monitored for the presence of the biomolecule. In otherembodiments the heterologous nucleic acid sequence is not expressed, butthe exogenous vector is cloned in the Vibrio sp. organism.Growth-conducive media support growth of the organism and examples areprovided herein.

In some aspects, the present disclosure provides an isolated orsynthesized nucleic acid molecule comprising SEQ ID NO:1 or a variantthereof. In some examples, the isolated or synthesized nucleic acidmolecule further comprises heterologous sequence on the 5′ and 3′ end,wherein the heterologous 5′ and 3′ sequences are compatible for cloninginto a target vector. Cloning can be performed by any known methods inthe art.

In some aspects, the present disclosure provides vectors comprisingVibrio sp. chromosomal replication machinery. In some examples, thereplication machinery comprises SEQ ID NO: 1 or a variant thereof. Insome examples, the vector further comprises a heterologous nucleic acidof interest. In some examples, the vector further comprises an induciblepromoter operably linked to a nucleic acid of interest. In someexamples, the vector is capable of replication in E. coli or S.cerevisiae.

In some embodiments, the present disclosure provides a vector comprisingthe replication machinery from a Vibrio sp. chromosome. In some aspects,the replication machinery is that of Chromosome II from Vibrio sp.bacteria. In some aspects, the replication machinery comprises SEQ IDNO: 1 or a variant thereof. In some aspects, the vector comprises aheterologous nucleic acid of interest. In some aspects, the nucleic acidof interest is at least 1 kb or at least 10 kb, or at least 25 kb, or atleast 50 kb, or at least 75 kb, or at least 100 kb, or at least 125 kb,or at least 150 kb, or at least 175 kb, or at least 200 kb, or at least250 kb, or at least 300 kb, or at least 350 kb, or at least 400 kb, orat least 500 kb, or at least 600 kb, or at least 700 kb, or at least 800kb, or at least 900 kb or at least 1 Mb, or 2 Mb, or 3 Mb, or 5 Mb, or 7Mb, or 10 kb-1 Mb or 25 kb-1 Mb or 50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2Mb or 50 kb-3 Mb or 50 kb-5 Mb or 50 kb-7 Mb, or 30 kb-1 Mb or 100 kb-1Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5 Mb or 30 kb-7 Mb or 100 kb-2Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100 kb-7 Mb. In some aspects, thevector can have a promoter, an origin of replication, an origin oftransfer, selectable marker, a counter selectable marker, a reportergene, a regulatory element, an enzyme gene, or any combination thereof,including that one or more of the elements may be omitted. In someaspects, the selectable marker is an antibiotic resistance gene, a geneencoding a polypeptide conferring resistance to a toxin, an auxotrophicmarker, or a combination thereof. In some aspects, the antibioticresistance gene confers resistance to antibiotics which is bleomycin,carbenicillin, chloramphenicol, gentamycin, glyphosate, hygromycin,kanamycin, neomycin, nourseothricin, phleomycin, puromycin,spectinomycin, streptomycin, or tetracyclin. In some examples, thereporter gene can be a fluorescent protein or beta-galactosidase. Insome aspects, the enzyme is a recombinase, integrase, nuclease,recombineering enzymes, or polymerase. In some examples the recombinaseis Cre recombinase. In some examples the integrase is PhiC31 or bxb1. Insome examples the nuclease is a TypeII CRISPR Cas9 nuclease. In someexamples, the polymerase is a Sp6, T3, or T7 RNA polymerase. In someaspects, the vector is compatible with E. coli, V. natriegens, and/or S.cerevisiae. In some aspects, the inducible promoter is induced by IPTG,arabinose, or temperature.

Any of the nucleic acids or vectors disclosed herein can utilize aninducible promoter. In addition to the inducible promoters describedherein, the inducible promoter can also be any chemically regulated,tetracycline regulated, steroid regulated, metal regulated orpathogenesis regulated promoter. Examples of chemically induciblepromoters include the AlcA promoter, which is induced by alcohol or aketone. Any of the promoters can also be inducible by physicalparameters, for example temperature-inducible or light inducible.

In some aspects, the present disclosure provides a compositioncomprising any of the genetically engineered Vibrio sp. disclosedherein. In some examples, the genetically engineered Vibrio sp. bacteriadisclosed herein are naturally competent. In some examples, thegenetically engineered Vibrio sp. bacteria are competent cells generatedby any of the methods disclosed herein. In some examples, the competentgenetically engineered Vibrio sp. bacteria are generated by the processof: (a) growing genetically modified Vibrio sp. bacterial cells in agrowth-conducive medium; (b) rendering said Vibrio sp. bacterial cellscompetent; and (c) freezing the cells.

In some aspects, the present disclosure provides an expression systemwhich comprises a vector comprising the Vibrio sp. chromosomalreplication machinery. In some examples, the replication machinerycomprises SEQ ID NO:1 or a variant thereof. In some examples, the vectorfurther comprises a heterologous nucleic acid of interest, such as anydescribed herein. In some examples, the vector further comprises aninducible promoter operably linked to a nucleic acid of interest. Insome examples, the vector is capable of replication in E. coli or S.cerevisiae. In some aspects, the vector further comprises a promoter, anorigin of replication, an origin of transfer, a selectable marker, acounter-selectable marker, a reporter gene, a regulatory element, anenzyme gene, or a combination thereof. In some aspects, selectablemarker is an antibiotic resistance gene, a gene encoding a polypeptideconferring resistance to a toxin, an auxotrophic marker, or anycombination thereof, including that one or more elements can be omitted.In some aspects, the antibiotic resistance gene confers resistance toantibiotics which is bleomycin, carbenicillin, chloramphenicol,gentamycin, glyphosate, hygromycin, kanamycin, neomycin, nourseothricin,phleomycin, puromycin, spectinomycin, streptomycin, or tetracyclin. Insome examples, the reporter gene can be a fluorescent protein orbeta-galactosidase. In some aspects, the enzyme is a recombinase,integrase, nuclease, recombineering enzymes, or polymerase. In someexamples the recombinase is Cre recombinase. In some examples theintegrase is PhiC31 or bxb1. In some examples the nuclease is a TypeIICRISPR Cas9 nuclease. In some examples, the polymerase is a Sp6, T3, orT7 RNA polymerase. In some aspects, the replication machinery comprisesSEQ ID NO: 1. In some aspects, the vector is compatible with E. coli, V.natriegens, and/or S. cerevisiae. In some aspects, the induciblepromoter is induced by IPTG, arabinose, or temperature. In some aspects,the vector further comprises a nucleic acid of interest. In someaspects, the polynucleotide of interest is at least 1 kb or at least 10kb, or at least 25 kb, or at least 50 kb, or at least 75 kb, or at least100 kb, or at least 125 kb, or at least 150 kb, or at least 175 kb, orat least 200 kb, or at least 250 kb, or at least 300 kb, or at least 350kb, or at least 400 kb, or at least 500 kb, or at least 600 kb, or atleast 700 kb, or at least 800 kb, or at least 900 kb or at least 1 Mb,or 2 Mb, or 3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1 Mb or 50kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5 Mb or 50kb-7 Mb, or 30 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5 Mb or 30kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100 kb-7 Mb.

In some aspects, the present disclosure provides host cells comprising avector comprising Vibrio sp. chromosomal replication machinery. In someexamples, the host cells are naturally competent. In some examples, thehost cells are competent cells generated by any of the herein disclosedmethods. In some examples, the vector is introduced into the host cellby transformation, transduction, biolistics, conjugation, chemicalcompetence, natural competence, or electroporation. In some examples,the replication machinery comprises SEQ ID NO: 1 or a variant thereof.In some examples, the vector further comprises a heterologous nucleicacid of interest. In some examples, the vector further comprises aninducible promoter operably linked to a nucleic acid of interest. Insome examples, the host cells are any genetically engineered Vibrio sp.bacteria disclosed herein. In some aspects, the vector further comprisesa promoter, an origin of replication, an origin of transfer, aselectable marker, a counter selectable marker, a reporter gene, aregulatory element, an enzyme gene, or a combination thereof. In someaspects, the selectable marker is an antibiotic resistance gene, a geneencoding a polypeptide conferring resistance to a toxin, an auxotrophicmarker, or a combination thereof. In some aspects, the antibioticresistance gene confers resistance to antibiotics which is bleomycin,carbenicillin, chloramphenicol, gentamycin, glyphosate, hygromycin,kanamycin, neomycin, nourseothricin, phleomycin, puromycin,spectinomycin, streptomycin, or tetracyclin. In some examples, thereporter gene can be a fluorescent protein or beta-galactosidase. Insome aspects, the enzyme is a recombinase, integrase, nuclease,recombineering enzymes, or polymerase. In some examples the recombinaseis Cre recombinase. In some examples the integrase is PhiC31 or bxb1. Insome examples the nuclease is a TypeII CRISPR Cas9 nuclease. In someexamples, the polymerase is a Sp6, T3, or T7 RNA polymerase. In someaspects, the inducible promoter is induced by IPTG, arabinose, ortemperature. In some aspects, the vector is alternatively oradditionally compatible with E. coli and/or S. cerevisiae. In someaspects, the vector further comprises a nucleic acid of interest. Insome aspects, the nucleic acid of interest is at least 1 kb or at least10 kb, or at least 25 kb, or at least 50 kb, or at least 75 kb, or atleast 100 kb, or at least 125 kb, or at least 150 kb, or at least 175kb, or at least 200 kb, or at least 250 kb, or at least 300 kb, or atleast 350 kb, or at least 400 kb, or at least 500 kb, or at least 600kb, or at least 700 kb, or at least 800 kb, or at least 900 kb or atleast 1 Mb, or 2 Mb, or 3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1Mb or 50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5Mb or 50 kb-7 Mb, or 30 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5Mb or 30 kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100kb-7 Mb.

In some aspects, the present disclosure provides a method of producing apolypeptide comprising: a) culturing cells comprising a vectorcomprising a Vibrio sp. chromosomal replication machinery and a nucleicacid encoding the polypeptide under conditions effective for theproduction of the polypeptide; and b) harvesting the polypeptide. Insome examples, the replication machinery comprises SEQ ID NO: 1 or avariant thereof. In some examples, the vector further comprises aninducible promoter operably linked to a nucleic acid encoding thepolypeptide. In some examples, the vector is capable of replication inE. coli or S. cerevisiae. In some examples, the cultured cells areVibrio sp. bacterial cells. In some examples, the Vibrio sp. cells areany of those disclosed herein. In some examples the cultured cells orVibrio sp. cells are naturally competent. In some examples, the culturedcells or Vibrio sp. cells are competent cells generated by any of themethods disclosed herein. In some examples, the vector is introducedinto the cultured cells or Vibrio sp. cells by conjugation, chemicalcompetence, natural competence, or electroporation. In some aspects, thevector further comprises a promoter, an origin of replication, an originof transfer, a selectable marker, a counter-selectable marker, areporter gene, a regulatory element, an enzyme gene, or a combinationthereof. In some aspects, the selectable marker is an antibioticresistance gene, a gene encoding a polypeptide conferring resistance toa toxin, an auxotrophic marker, and a combination thereof. In someaspects, the antibiotic resistance gene confers resistance toantibiotics selected from the group consisting of bleomycin,carbenicillin, chloramphenicol, gentamycin, glyphosate, hygromycin,kanamycin, neomycin, nourseothricin, phleomycin, puromycin,spectinomycin, streptomycin, or tetracyclin. In some examples, thereporter gene can be a fluorescent protein or beta-galactosidase. Insome aspects, the enzyme is a recombinase, integrase, nuclease,recombineering enzymes, or polymerase. In some examples the recombinaseis Cre recombinase. In some examples the integrase is PhiC31 or bxb1. Insome examples the nuclease is a TypeII CRISPR Cas9 nuclease. In someexamples, the polymerase is a Sp6, T3, or T7 RNA polymerase. In someaspects, the vector is compatible with E. coli, V. natriegens, and/or S.cerevisiae. In some aspects, the inducible promoter is induced by IPTG,arabinose, or temperature. In some aspects, the nucleic acid is at least1 kb or at least 10 kb, or at least 25 kb, or at least 50 kb, or atleast 75 kb, or at least 100 kb, or at least 125 kb, or at least 150 kb,or at least 175 kb, or at least 200 kb, or at least 250 kb, or at least300 kb, or at least 350 kb, or at least 400 kb, or at least 500 kb, orat least 600 kb, or at least 700 kb, or at least 800 kb, or at least 900kb or at least 1 Mb, or 2 Mb, or 3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mbor 25 kb-1 Mb or 50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mbor 50 kb-5 Mb or 50 kb-7 Mb, or 30 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mbor 30 kb-5 Mb or 30 kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mbor 100 kb-7 Mb.

In some aspects, the present disclosure provides a method of producing apolypeptide comprising: a) contacting Vibrio sp. bacteria with a vectorcomprising a nucleic acid encoding the polypeptide and an induciblepromoter, such that the vector is introduced into the bacteria; b)growing the bacteria under conditions effective for production of thepolypeptide; and c) harvesting the polypeptide. In some examples, thevector comprises Vibrio sp. chromosomal replication machinery. In someexamples, the replication machinery comprises SEQ ID NO: 1 or a variantthereof. In some examples, the vector is capable of replication in E.coli or S. cerevisiae. In some examples, the Vibrio sp. cells are any ofthose disclosed herein. In some examples, the Vibrio sp. bacteria arenaturally competent. In some examples the Vibrio sp. bacteria arecompetent cells generated by any of the methods disclosed herein. Insome examples, the vector is introduced into the Vibrio sp. bacteria byconjugation, chemical competence, natural competence, orelectroporation. In some aspects, the vector further comprises apromoter, an origin of replication, an origin of transfer, a selectablemarker, a counter-selectable marker, a reporter gene, a regulatoryelement, an enzyme gene, or a combination thereof. In some aspects, theselectable marker is an antibiotic resistance gene, a gene encoding apolypeptide conferring resistance to a toxin, an auxotrophic marker, ora combination thereof. In some aspects, the antibiotic resistance geneconfers resistance to antibiotics which is bleomycin, carbenicillin,chloramphenicol, gentamycin, glyphosate, hygromycin, kanamycin,neomycin, nourseothricin, phleomycin, puromycin, spectinomycin,streptomycin, and tetracyclin. In some examples, the reporter gene canbe a fluorescent protein or beta-galactosidase. In some aspects, theenzyme is a recombinase, integrase, nuclease, recombineering enzymes, orpolymerase. In some examples the recombinase is Cre recombinase. In someexamples, the integrase is PhiC31 or bxb1. In some examples the nucleaseis a TypeII CRISPR Cas9 nuclease. In some examples, the polymerase is aSp6, T3, or T7 RNA polymerase. In some aspects, the inducible promoteris induced by IPTG, arabinose, or temperature. In some aspects, thenucleic acid is at least 1 kb or at least 10 kb, or at least 25 kb, orat least 50 kb, or at least 75 kb, or at least 100 kb, or at least 125kb, or at least 150 kb, or at least 175 kb, or at least 200 kb, or atleast 250 kb, or at least 300 kb, or at least 350 kb, or at least 400kb, or at least 500 kb, or at least 600 kb, or at least 700 kb, or atleast 800 kb, or at least 900 kb or at least 1 Mb, or 2 Mb, or 3 Mb, or5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1 Mb or 50 kb-1 Mb or 100 kb-1 Mbor 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5 Mb or 50 kb-7 Mb, or 30 kb-1 Mbor 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5 Mb or 30 kb-7 Mb or 100 kb-2 Mbor 100 kb-3 Mb or 100 kb-5 Mb or 100 kb-7 Mb. In some aspects, theVibrio sp. cells comprise one or two altered, rearranged, or minimizedchromosomes. In some examples, the essential elements from Chromosome IIare alternatively located on Chromosome I. In some examples, theengineered bacteria contain a single chromosome comprising the essentialfeatures of Chromosome I and II. In some aspects, the growth doublingtime of the cells is about 5 minutes to 15 minutes.

In some aspects, the present disclosure provides a method for cloning anucleic acid comprising: a) introducing a heterologous nucleic acid intoVibrio sp. bacteria to create a transformed bacteria; b) culturing thecells under conditions for growth of the cells; c) isolation of a singletransformed bacterial colony; d) growth of the bacterial colony; and e)extraction of nucleic acid. In some examples, the Vibrio sp. bacteriaare those of any of those disclosed herein. In some examples the Vibriosp. bacteria are naturally competent. In some examples, the Vibrio sp.bacteria are competent cells generated by any of the methods disclosedherein. In some examples, the introduction of the nucleic acid isperformed by conjugation, chemical competence, natural competence, orelectroporation. In some examples, the heterologous nucleic acid is avector. In some examples, the vector comprises Vibrio sp. chromosomalreplication machinery. In some examples, the replication machinerycomprises SEQ ID NO:1 or a variant thereof. In some examples, the vectoris capable of replication in E. coli or S. cerevisiae. In some aspects,the introduction of the nucleic acid is performed by conjugation,transformation, transduction, chemical competence, natural competence,or biolistics. In some aspects, the nucleic acid is at least 1 kb or atleast 10 kb, or at least 25 kb, or at least 50 kb, or at least 75 kb, orat least 100 kb, or at least 125 kb, or at least 150 kb, or at least 175kb, or at least 200 kb, or at least 250 kb, or at least 300 kb, or atleast 350 kb, or at least 400 kb, or at least 500 kb, or at least 600kb, or at least 700 kb, or at least 800 kb, or at least 900 kb or atleast 1 Mb, or 2 Mb, or 3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1Mb or 50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5Mb or 50 kb-7 Mb, or 30 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5Mb or 30 kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100kb-7 Mb.

In some aspects, the present disclosure provides a kit for cloning DNAcomprising: a) a vector comprising the Vibrio sp. chromosomalreplication machinery; b) host cells compatible with the vector; c)buffer compatible with the host cells; and d) instructions for cloningthe DNA. In some examples, the vector further comprises an induciblepromoter. In some examples, the replication machinery comprises SEQ IDNO: 1 or a variant thereof. In some examples, the host cells are Vibriosp. bacteria. In some examples the Vibrio sp. bacteria are any of thosedisclosed herein. In some examples, the host cells are E. coli or S.cerevisiae. In some aspects, the vector further comprises a promoter, anorigin of replication, an origin of transfer, a selectable marker, acounter-selectable marker, a reporter gene, a regulatory element, anenzyme gene, or a combination thereof. In some aspects, the selectablemarker is an antibiotic resistance gene, a gene encoding a polypeptideconferring resistance to a toxin, an auxotrophic marker, and acombination thereof. In some aspects, the antibiotic resistance geneconfers resistance to antibiotics which is bleomycin, carbenicillin,chloramphenicol, gentamycin, glyphosate, hygromycin, kanamycin,neomycin, nourseothricin, phleomycin, puromycin, spectinomycin,streptomycin, and tetracyclin. In some examples, the reporter gene canbe a fluorescent protein or beta-galactosidase. In some aspects, theenzyme is a recombinase, integrase, nuclease, recombineering enzymes, orpolymerase. In some examples the recombinase is Cre recombinase. In someexamples the integrase is PhiC31 or bxb1. In some examples the nucleaseis a TypeII CRISPR Cas9 nuclease. In some examples, the polymerase is aSp6, T3, or T7 RNA polymerase. In some aspects, the inducible promoteris induced by IPTG, arabinose, or temperature. In some aspects, thevector is alternatively or additionally compatible with E. coli and/orS. cerevisiae. In some aspects, the nucleic acid is at least 1 kb or atleast 10 kb, or at least 25 kb, or at least 50 kb, or at least 75 kb, orat least 100 kb, or at least 125 kb, or at least 150 kb, or at least 175kb, or at least 200 kb, or at least 250 kb, or at least 300 kb, or atleast 350 kb, or at least 400 kb, or at least 500 kb, or at least 600kb, or at least 700 kb, or at least 800 kb, or at least 900 kb or atleast 1 Mb, or 2 Mb, or 3 Mb, or 5 Mb, or 7 Mb, or 10 kb-1 Mb or 25 kb-1Mb or 50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5Mb or 50 kb-7 Mb, or 30 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or 30 kb-5Mb or 30 kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or 100kb-7 Mb.

In some aspects, the present disclosure provides a kit comprisingcompetent Vibrio sp. bacterial cells, which can be any disclosed herein.In some examples, the kit further comprises a compatible cloning vector.

Alternatively, or in addition to any of the forgoing embodiments, thedisclosure provides the following embodiments:

Embodiment 1 is genetically engineered Vibrio sp. bacteria comprisingone or more of the following:

-   -   a) altered Chromosome I or Chromosome II,    -   b) one or more non-essential genes removed from either        Chromosome I or Chromosome II,    -   c) one or more genes removed that encode an element selected        from the group consisting of an endonuclease, exonuclease,        methylase, nuclease, restriction enzyme, and        restriction-modification system,    -   d) at least one essential element from Chromosome II        alternatively located on an engineered Chromosome I,    -   e) at least one essential element from Chromosome II        alternatively located on an engineered Chromosome I, wherein the        essential element is a gene required for a function selected        from the group consisting of metabolism, DNA replication,        transcription, translation, cellular structure maintenance, and        transport processes into and out of the cell,    -   f) a single chromosome comprising essential elements from        Chromosome I and Chromosome II such that the single chromosome        is capable of supporting survival and replication of the        bacterial under non-selective conditions,    -   g) a heterologous nucleic acid sequence operably linked to a        heterologous promoter,    -   h) a heterologous nucleic acid encoding T7 RNA polymerase        operably linked to an inducible promoter, or    -   i) natural or lab-generated competence.

Embodiment 2 is a method of producing competent Vibrio sp. cellscomprising a) growing Vibrio sp. cells in a growth-conducive medium, b)rendering said Vibrio sp. cells competent, and c) freezing the cells.

Embodiment 3 is a method of producing a biomolecule comprising a)contacting Vibrio sp. bacteria with a heterologous nucleic acid encodingthe biomolecule, such that the heterologous nucleic acid is introducedinto the bacteria, b) growing the bacteria in a growth-conducive mediumwherein the heterologous nucleic acid is expressed, thereby producingthe biomolecule, and c) isolating the biomolecule.

Embodiment 4 is a Vibrio sp. chromosomal replication machinery.

Embodiment 5 is the Vibrio sp. replication machinery comprising SEQ IDNO:1.

Embodiment 6 is a vector comprising Embodiment 4 or Embodiment 5 andoptionally further comprising: a) a heterologous nucleic acid ofinterest optionally operably linked to an inducible promoter, b)replication machinery compatible with E. coli or S. cerevisiae.

Embodiment 7 is an expression system comprising the vector of Embodiment6.

Embodiment 8 is a host cell comprising the vector of Embodiment 6,wherein the host cells are optionally any of those from Embodiment 1.

Embodiment 9 is a method of producing a polypeptide comprising a)culturing cells comprising a vector comprising a Vibrio sp. chromosomalreplication machinery and a nucleic acid encoding the polypeptide underconditions effective for the production of the polypeptide; and b)harvesting the polypeptide.

Embodiment 10 is a method of producing a polypeptide comprising a)contacting Vibrio sp. bacteria with a vector comprising a nucleic acidencoding the polypeptide and an inducible promoter, such that the vectoris introduced into the bacteria; b) growing the bacteria underconditions effective for production of the polypeptide; and c)harvesting the polypeptide.

Embodiment 11 is a method of cloning a nucleic acid comprising a)introducing a heterologous nucleic acid into Vibrio sp. bacteria tocreate a transformed bacteria; b) culturing the cells under conditionsfor growth of the cells; c) isolation of a single transformed bacterialcolony; d) growth of the bacterial colony; and e) extraction of nucleicacid.

Embodiment 12 is the methods of any of Embodiments 2, 3, 9, 10, or 11wherein the cells are those of Embodiment 1.

Embodiment 13 is the method of any of Embodiments 3, 9, 10, 11 whereinthe vector is that of Embodiment 6 or the heterologous nucleic acidcomprises Embodiment 4 or Embodiment 5.

Embodiment 14 is a kit for cloning DNA comprising a) a vector comprisingthe Vibrio sp. chromosomal replication machinery, b) host cellscompatible with the vector, c) buffer compatible with the host cells,and d) instructions for cloning the DNA.

Embodiment 15 is the kit of Embodiment 14, wherein the vector is that ofEmbodiment 6.

Embodiment 16 is a kit comprising competent Vibrio sp. cells.

Embodiment 17 is the kit of Embodiment 14 or 16, wherein the cells areany of those of Embodiment 1.

Embodiment 18 is that of any of Embodiments 1-17, wherein Vibrio sp. ispreferably Vibrio natriegens.

Vector

The present invention also discloses vectors operable in a Vibrio sp.The vectors can be a cloning, shuttle, or expression vectors (orcombination thereof) depending on features included. Any of the vectorscan have a sequence from Vibrio sp. chromosome II that comprises SEQ IDNO: 1 or a variation thereof. Any of the vectors disclosed herein canhave a sequence of at least 2 kb or at least 3 kb or at least 4 kb or atleast 5 kb of SEQ ID NO: 1, or a variant of any of them. The vector canalso have an origin of replication operable in Vibrio sp. In someembodiments the origin of replication can be, for example, a p15aorigin, or the origin of replication from plasmid pBR325 or SEQ ID NO:2. But the person of ordinary skill with reference to this disclosurewill realize other origins of replication that will find use in theinvention. The vector can also have a selectable marker (e.g., anantibiotic resistance gene), and a heterologous nucleic acid ofinterest, for example a DNA sequence that encodes a heterologous proteinor peptide, as described herein. The heterologous DNA sequence can be atleast 5 kb or at least 10 kb or at least 25 kb or at least 50 kb or atleast 100 kb or at least 125 kb or at least 150 kb or at least 175 kb orat least 200 kb or at least 250 kb, or at least 300 kb, or at least 350kb, or at least 400 kb, or at least 500 kb, or at least 600 kb, or atleast 700 kb, or at least 800 kb, or at least 900 kb or at least 1 Mb,or at least 2 Mb, or at least 3 Mb, or at least 5 Mb, or at least 7 Mb,or 10-100 kb or 10-150 kb or 10-200 kb or 10-500 kb or 10-700 kb or10-1000 kb or 10 kb-2 Mb or 10 kb-5 Mb, or 10 kb-1 Mb or 25 kb-1 Mb or50 kb-1 Mb or 100 kb-1 Mb or 50 kb-2 Mb or 50 kb-3 Mb or 50 kb-5 Mb or50 kb-7 Mb, or 30 kb-1 Mb or 100 kb-1 Mb or 30 kb-2 Mb or 30 kb-3 Mb or30 kb-5 Mb or 30 kb-7 Mb or 100 kb-2 Mb or 100 kb-3 Mb or 100 kb-5 Mb or100 kb-7 Mb. or any length as disclosed herein. In some embodiments theheterologous sequence can encode a valuable protein or polypeptide,e.g., an enzyme, an immunoglobulin (e.g., trastuzumab, eculizumab,natalizumab, cetuximab, omalizumab, usteinumab, panitumumab, adalimumab,or a functional fragment of any of them), pro-insulin, or insulin.

The vector can also have an origin of replication operable in a secondspecies of organism, which can be a non-Vibrio and non-E. coli organism.The second species can be another bacteria, for example, a Bacillus sp.The vector can therefore be a shuttle vector. The vector can also beoperable in a yeast (e.g., % a yeast of the genus Saccharomyces, S.cerevisiae, S. pombe), in addition to the Vibrio sp. and the non-Vibrioand non-E. coli species of organism.

The vector can also have one or two or three or more than three operablepromoters. Examples include, but are not limited to, the IPTG-inducibletrc promoter, the arabinose-inducible araBAD or araC promoters, and thetemperature-inducible lambda pR promoter modulated by thetemperature-sensitive cI857 repressor. In one embodiment theheterologous DNA sequence is under the control of an inducible promoter.One or more promoters can regulate the heterologous DNA sequence.

The vectors of the invention can also have a transcriptional terminator,which can follow a heterologous DNA sequence that encodes a heterologousprotein. One example of suitable transcriptional terminators includesthe rrnB transcriptional termination sequence.

By “operable” is meant that the vector is effective for the expressionof heterologous DNA sequences on the vector, or is effective for cloningof the vector and/or heterologous DNA sequences on the vector. When theheterologous DNA sequence encodes a heterologous protein, it istranslated into a protein or polypeptide molecule and is produced as afunctional protein or polypeptide in detectable amounts that aresufficient to provide the relevant function.

The vector can also comprise one or more selectable marker(s) (e.g.,resistance gene(s)). The selectable marker(s) can be any suitable markerand examples include, but are not limited to, tetA/tetR,chloramphenicol, Trp, or any described herein. Selectable markers can bespecific for the respective organisms when a shuttle vector is employed.

The vector can also encode a heterologous DNA sequence, which in someembodiments encodes a gene of interest (goi). The heterologous DNAsequence can encode a protein or polypeptide, which can be expressedfrom the shuttle vector to produce a functional protein or polypeptide.The functional protein or polypeptide can be any protein or polypeptide,for example an enzyme, an immunoglobulin, or any protein of interest, asdescribed herein.

The vector can also be a shuttle vector and also have an origin oftransfer (oriT) for conjugal transfer of the shuttle vector from theVibrio sp. to the second species of organism. The transfer can beRP4-mediated conjugal transfer between the Vibrio sp. and the secondspecies of organism. The vector can also have an autonomouslyreplicating sequence (ARS) and a yeast centromere sequence (CEN) forreplication in a yeast, such as described herein. The vector can alsohave a multiple cloning site (MCS) that has at least two restrictionrecognition sites such as, for example, sites for EcoRI, BamHI, or Pstl.But sites for any suitable restriction enzymes can be employed. Thevector can also have a selection marker or resistance gene for a yeast,for example a Trp sequence or another suitable selection marker orresistance gene.

Any of the vectors described herein, including the shuttle vectors, canbe low copy number vectors or plasmids, as maintained in Vibrio sp.organisms of the invention. In various embodiments the plasmids or(shuttle) vectors can have a copy number of less than 10 or less than 9or less than 8 or less than 7 or less than 6 or less than 5. Any of thevectors of the invention can also have one or more or two or more loxsites (e.g., loxP sequence derived from bacteriophage P1). In any of theembodiments disclosed herein the Vibrio sp. organism can be a Vibrionatriegens. The invention also provides any Vibrio sp. organismdescribed herein that comprises any vector or nucleic acid describedherein.

Vibrio sp. Strain

The invention also provides a Vibrio sp. organism having specificcharacteristics. The Vibrio sp. organism can have a nucleic acidsequence encoding a T7 RNA polymerase that is regulated by an induciblepromoter. The inducible promoter can be a lac promoter that is induciblewith IPTG, for example the native lac promoter or the lacUV5 promoter.In other embodiments the inducible promoter can be the araBAD or araCpromoter, which are inducible with arabinose. But in other embodimentsthe inducible promoter can be any suitable promoter inducible with aconvenient metabolite. The Vibrio sp. organism can also have a ladrepressor for repression of the inducible promoter. The organism canalso have a nucleic acid sequence encoding a heterologous protein orpeptide regulated by the inducible promoter and, optionally, a modifiedor weakened ribosome binding site. The sequence encoding the T7 RNApolymerase and inducible promoter can be integrated into Chromosome I orII of the organism, or present on a vector. FIG. 1 provides severalexamples of vectors that can be constructed according to the invention.The components of the vectors are not limited to the specificarrangements and can be used in any suitable combination. In somespecific examples the Vibrio sp. can have a deletion of a nuclease,which can be an extracellular nuclease (e.g., a Dns deletion), a lacUV5promoter regulating a T7 RNA polymerase gene with a lad repressor; andoptionally can have SEQ ID NO: 8 or a variant thereof as a weakened rbs;all of which can be present on either Chromosome I or II, or on a vectorwithin the organism. In another example the organism can use the araBADand/or araC promoter 5′ to the T7 RNA polymerase gene, and optionallythe weakened ribosome binding site (e.g., SEQ ID NO: 8 or a variantthereof). These sequences also can be present on Chromosome I or II, oron a vector within the organism.

In any of the organisms or vectors a sequence encoding a heterologousprotein or peptide can also contain a sequence that codes for asecretion signal for the protein or peptide, allowing the protein orpeptide to be secreted from the cell after synthesis. The Vibrio sp.organism can be a Vibrio natriegens. Several examples of signalsequences operable in Vibrio sp. are provided as SEQ ID Nos: 11-25, orvariants of any of them.

Ribosome Binding Site

Any of the Vibrio sp. organisms of the invention can also have amodified or weakened regulatory element, such as a ribosome binding site(rbs). The rbs can be present on Chromosome I or II of the organism. Theorganism can also have a nucleic acid sequence encoding a T7 RNApolymerase, and an optional inducible promoter. The rbs and induciblepromoter (when present) can be present in front of (5′ to) the T7 RNApolymerase gene. The rbs can be modified to be a weaker binder ofribosomes. Thus, when RNA is transcribed from the gene it is weaklybound by the modified rbs and therefore less protein is produced, andthe rbs can therefore regulate expression of the gene through lesstranslation of encoded protein or polypeptide. The modified or weakenedrbs can reduce background levels of protein expressed from the regulatedgene. Background levels of a protein is the level of protein expressionthat occurs when a native ribosome and inducible promoter are present,and the inducer is not present. The modified or weakened rbs can resultin background protein expression from the regulated gene at least 25%less or at least 30% less or at least 40% less or at least 50% less thanthe regulated gene with a native rbs and the same inducible promoter.Background expression can be easily measured, for example usingdensitometry on a PAGE gel. The Vibrio sp. of the invention can alsohave at least one deletion of a nuclease, e.g., deletion of anextracellular nuclease such as a Dns deletion. In one embodiment themodified or weakened rbs can have the sequence of SEQ ID NO: 8 or avariant thereof. Variants of SEQ ID NO: 8 can be as described herein,and can have one or two or three or four or five substitutionmodifications, which can include nucleotide substitutions, deletions orinsertions. The rbs can be modified or “weakened” so that ribosomes bindless strongly, and therefore less background gene expression occurs.

Thus, in one embodiment the invention provides a Vibrio sp. organismthat has a genome having an exogenous gene for a T7 RNA polymerase thatis regulated by an inducible promoter. The organism can also have amodified rbs that regulates expression of the T7 RNA polymerase gene byregulating translation of the RNA gene product. The organism can alsohave an exogenous gene that encodes a heterologous DNA sequence asdisclosed herein (e.g., encodes a heterologous protein) that can beregulated by the inducible promoter. The exogenous gene for a T7 RNApolymerase and inducible promoter can be integrated into chromosome I orII of the organism. The heterologous DNA sequence can also be onchromosome I or II of the organism, or can be contained on a plasmid orother vector comprised in the organism. Any of the inducible promotersdescribed herein can be utilized in the invention (e.g., araBAD, lac,lacUV5). The gene for a T7 RNA polymerase can also have a repressorsequence for repression of the inducible promoter (e.g., lad).

EXAMPLES

The disclosure in all its aspects is illustrated further in thefollowing Examples. The Examples do not, however, limit the scope of thedisclosure, which is defined by the appended claims.

Example 1 Growth of V. natriegens in a Range of Growth Media as Well asat Multiple Temperatures

Growth of V. natriegens was examined on a number of different growthmedia and at multiple temperatures. A glycerol stock of V. natriegenswas used to inoculate liquid cultures or was streaked out on agarplates. Liquid cultures were cultivated with agitation ranging from175-220 RPM at the indicated temperatures. After overnight incubation,plates/cultures were examined for growth. Growth was defined asturbidity (in the case of liquid cultures) or visible colonies (in thecase of agar plates).

Media compositions are as follows:

LB broth: 10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl.

LB broth+v2 salts: LB broth supplemented with additional salts (204 mMNaCl, 4.2 mM KCl, and 23.14 mM MgCl2).

LB broth+v2 salts+glucose: LB broth supplemented with additional salts(204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)+0.2% glucose.

LB broth+v3 salts: LB broth supplemented with additional salts (475 mMNaCl, 9.7 mM KCl, and 54 mM MgCl2).

LB broth+v3 salts+glucose: LB broth supplemented with additional salts(475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)+0.2% glucose.

LB agar: LB media+1.5% agar-agar.

LB agar+v2 salts: LB broth supplemented with additional salts (204 mMNaCl, 4.2 mM KCl, and 23.14 mM MgCl2)+1.5% agar-agar.

LB agar minus NaCl with 6% sucrose: 10.0 g/L Tryptone, 5.0 g/L YeastExtract, 1.5% agar-agar, 6% sucrose.

Nutrient Broth+1.5% NaCl: 8 g/L Difco™ Nutrient Broth (Cat. No. 234000)supplemented with 1.5% NaCl.

Nutrient Agar+1.5% NaCl: 8 g/L Difco™ Nutrient Broth (Cat. No. 234000)supplemented with 1.5% NaCl and 1.5% agar-agar.

Brain Heart Infusion Broth: 37 g/L Teknova Brain Heart Infusion BrothDry Media (Cat. No. B9500).

Brain Heart Infusion Broth+2% NaCl: 37 g/L Teknova Brain Heart InfusionBroth Dry Media (Cat. No. B9500)+20 g/L NaCl.

Brain Heart Infusion Broth+1.5% Instant Ocean: 37 g/L Teknova BrainHeart Infusion Broth Dry Media (Cat. No. B9500)+15 g/L Instant Ocean SeaSalt Mixture.

Brain Heart Infusion Broth+v2 salts: 37 g/L Teknova Brain Heart InfusionBroth Dry Media (Cat. No. B9500) supplemented with additional salts (204mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2).

Brain Heart Infusion Broth+v3 salts: 37 g/L Teknova Brain Heart InfusionBroth Dry Media (Cat. No. B9500) supplemented with additional salts (475mM NaCl, 9.7 mM KCl, and 54 mM MgCl2).

Brain Heart Infusion Agar+1.5% Instant Ocean: 52 g/L Difco™ Brain HeartInfusion Agar (Cat. No. 241830)+15 g/L Instant Ocean Sea Salt Mixture.

Brain Heart Infusion Agar: 37 g/L Teknova Brain Heart Infusion Broth DryMedia (Cat. No. B9500)+1.5% agar-agar.

Brain Heart Infusion Agar+v2 salts: 37 g/L Teknova Brain Heart InfusionBroth Dry Media (Cat. No. B9500)+1.5% agar-agar supplemented withadditional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2).

M9 glucose media (500 mL): 1× M9 Salts, 0.4% glucose, 2 mM MgSO4, 0.1 mMCaCl₂.

M9 glucose agar: M9 glucose media supplemented with 1.5% agar-agar.

M9 glucose with 1% sucrose: 1×M9 Salts, 0.4% glucose, 2 mM MgSO4, 0.1 mMCaCl₂, 1% sucrose.

M9 glucose with 2% sucrose: 1×M9 Salts, 0.4% glucose, 2 mM MgSO4, 0.1 mMCaCl₂, 2% sucrose.

M9 glucose with 4% sucrose: 1×M9 Salts, 0.4% glucose, 2 mM MgSO4, 0.1 mMCaCl₂, 4% sucrose.

M9 1% sucrose: 1×M9 Salts, 2 mM MgSO4, 0.1 mM CaCl₂, 1% sucrose.

M9 2% sucrose: 1×M9 Salts, 2 mM MgSO4, 0.1 mM CaCl₂, 2% sucrose.

M9 4% sucrose: 1×M9 Salts, 2 mM MgSO4, 0.1 mM CaCl₂, 4% sucrose.

marine agar: 55.1 g/L Difco™ Marine Agar 2216 (Cat. No. 212185).

Bacto Heart Infusion Broth: 25 g/L Bacto™ Heart Infusion Broth (Cat. No.238400).

SSG agar: 28 g/L Bacto™SOB Medium (Cat. No. 244310), 17% Fetal BovineSerum, 1% glucose, 4 mL/L Phenol Red Solution (Sigma P0290).

2xYT+v2 salts+glucose+phosphate buffer: 2xYT media (16 g/L Tryptone, 10g/L Yeast Extract, 5 g/L NaCl) is supplemented with v2 salts (204 mMNaCl, 4.2 mM KCl, 23.14 mM MgCl₂), 17.61 mM Na₂HPO₄, 0.2% glucose. pH isadjusted to 7.4.

Vegitone Infusion Broth+v2 salts: Vegitone Infusion Broth (Sigma Aldrichcat #41960) supplemented with v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl₂).

LB+v2 salts+glucose+phosphate buffer: LB media (10 g/L Tryptone, 5 g/LYeast Extract, 10 g/L NaCl) is supplemented with v2 salts (204 mM NaCl,4.2 mM KCl, 23.14 mM MgCl₂), 17.6 mM K₂HPO₄, 0.2% glucose. pH isadjusted to 7.0.

Results of the growth experiments are presented in Table 1:

TABLE 1 Media Format 25° C. 30° C. 37° C. LB broth liquid Growth growthno growth culture LB broth + v2 salts liquid N/A growth growth cultureLB broth + v2 salts + liquid N/A growth growth glucose culture LBbroth + v3 salts liquid N/A growth growth culture LB broth + v3 salts +liquid N/A growth growth glucose culture LB agar agar plate Faint growthgrowth LB agar + v2 salts agar plate N/A growth growth LB agar minusNaCl agar plate N/A no growth no growth with 6% sucrose Nutrient Broth +liquid Growth growth no growth 1.5% NaCl culture Nutrient Agar + agarplate slow growth attenuated 1.5% NaCl growth growth Brain HeartInfusion liquid Growth growth attenuated Broth culture growth BrainHeart Infusion liquid Growth growth growth Broth + 2% NaCl culture BrainHeart Infusion liquid Growth growth growth Broth + 1.5% Instant cultureOcean Brain Heart Infusion liquid N/A growth growth Broth + v2 saltsculture Brain Heart Infusion liquid N/A growth growth Broth + v3 saltsculture Brain Heart Infusion agar plate slow growth growth Agar + 1.5%Instant growth Ocean Brain Heart Infusion agar plate N/A growth N/A agarBrain Heart Infusion agar plate Growth growth growth agar + v2 salts M9glucose media liquid slow growth N/A culture growth M9 glucose agar agarplate slow growth slow growth growth M9 glucose with 1% agar plate N/Agrowth growth sucrose M9 glucose with 2% agar plate N/A growth growthsucrose M9 glucose with 4% agar plate N/A growth growth sucrose M9 1%sucrose agar plate N/A growth growth M9 2% sucrose agar plate N/A growthgrowth M9 4% sucrose agar plate N/A growth growth marine agar agar plateslow growth growth growth Bacto Heart Infusion liquid N/A growthattenuated Broth culture growth SSG agar agar plate Growth growth growth2xYT + v2 Liquid N/A growth growth salts + glucose + culture phosphatebuffer Vegitone Infusion Liquid N/A growth growth Broth + v2 saltsculture LB + v2 salts + Liquid N/A growth growth glucose + phosphateculture buffer

Example 2 Transformation of V. natriegens with Exogenous DNA ConstructsVia Conjugation

This method was used to transfer a mobilizable plasmid from E. coli intoV. natriegens where:

1) the plasmid was maintained as an episomal molecule in V. natriegens,or

2) where (with appropriate plasmid design) the plasmid integrated intothe V. natriegens chromosome via a single or double-crossoverintegration event.

Donor Preparation: 10 mL of LB medium containing appropriate antibioticwas inoculated with E. coli donor strain (containing mobilizable plasmidof interest) and incubated overnight at 37° C. with agitation (200 RPM).Acceptable donor strains include, but are not limited to strain S17-1λpir (containing the RP4 conjugation machinery integrated into thechromosome) or EPI300 cells harboring the pRL443 conjugative plasmid.

Recipient Preparation: 10 mL of LB medium was inoculated with V.natriegens recipient strain and incubated overnight at room temperaturewith agitation (175 RPM).

Conjugative Mating: Donor and recipient cultures were retrieved fromincubators. 1 mL of each culture was separately centrifuged at 5000×gfor 3 min in a 1.5 mL Eppendorf tube to pellet the cells. Thesupernatant was decanted and the cell pellets were each resuspended in 1mL fresh LB medium. The wash (centrifugation/decanting/resuspension) wasrepeated for the donor strain to further reduce residual antibioticcarryover. Donor and recipient cultures were then mixed in multipledifferent ratios (e.g., 1:9, 1:4, 1:1, 4:1, 9:1 donor:recipient) in atotal volume of 100 μL. The 100 μL of cells were spotted out as 10 μLspots on prewarmed LB plates, and incubated at 30° C. for 3-5 hours.Cells were washed from plate with 1 mL M9 glucose medium. Variousvolumes of cells (1 μL, 5 μL, 20 μL) were plated out on M9 glucoseplates containing appropriate antibiotic and incubated overnight at 30°C. The E. coli donor strains mentioned above will not grow on the M9medium utilized for this procedure (see recipe below). Individual V.natriegens colonies that grew on the M9 selective plate were thenscreened for successful conjugation event via standard methods.

M9 glucose medium (500 mL):

-   -   100 mL 5×M9 Salts    -   390 mL ddH2O    -   7.5 g agar-agar*    -   10 mL 20% glucose**    -   1 mL 1 M MgSO4**    -   50 μL 1 M CaCl2**    -   for solid media, add agar-agar    -   *added after autoclaving

Example 3 Transformation of V. natriegens with Exogenous DNA ConstructsVia Electroporation

Preparation of Electrocompetent cells: 10 mL of Brain Heart InfusionBroth supplemented with supplemented salts (204 mM NaCl, 4.2 mM KCl, and23.14 mM MgCl2) was inoculated with Vibrio natriegens and incubatedovernight at 30° C. with agitation. On the following day, 250-500 mL ofthe same growth media was inoculated with the overnight culture at adilution of 1:100 to 1:200 (overnight culture:fresh media). The culturewas grown at 37° C. with shaking until an OD600 of 0.5. The culture wasthen split into two pre-chilled 250 mL centrifuge bottles which werethen incubated on ice for 0-20 min. The cells were pelleted at 6500 RPMin a JA-14 centrifuge rotor for 20 min at 4° C. The supernatant wascarefully decanted and the cell pellets were gently resuspended in 5-10mL of electroporation buffer (680 mM sucrose, 7 mM K₂HPO₄, pH 7). Thesuspension was transferred to centrifuge tubes and the tube was filledto top (˜35 mL) with additional electroporation buffer and invertedseveral times to mix. The cells were spun down at 6750 RPM for 15 min at4° C. in a JA-17 rotor. The supernatant was decanted with pipette. Thewash was repeated two times for a total of three washes. After the finalwash, the cells were gently resuspended in residual electroporationbuffer. The volume was adjusted with additional electroporation bufferto bring the final OD600 to 16. Cells were aliquoted into pre-chilledtubes and were stored at −80° C. until use.

Electroporation protocol: A vial of competent cells was retrieved fromstorage at −80° C. and allowed to thaw on ice. Plasmid DNA andelectrocompetent cells were combined and gently mixed in a pre-chilled1.5 mL microfuge tube. The cell/DNA suspension was transferred to apre-chilled electroporation cuvette with a 0.1 cm gap size. Cells wereelectroporated with the following parameters: 700-900 V, 25 μF, 200Ω, 1mm cuvette. Cells were immediately recovered in 500 μL recovery media(Brain Heart Infusion Broth supplemented with supplemented salts (204 mMNaCl, 4.2 mM KCl, 23.14 mM MgCl2, and 0-680 mM sucrose) and transferredto a 15 mL culture tube. The cells were recovered by incubating at30-37° C. for 1-2 hours. Aliquots of the recovery media were plated outon pre-warmed agar plates containing appropriate antibiotic. Acceptableagar media include, but are not limited to: M9 glucose, Brain HeartInfusion Agar (with or without additional salt supplementation), and LB(with our without additional salt supplementation). The plates wereincubated for several hours to overnight at 30-37° C. for colonies toappear.

Example 4 The Use of V. natriegens as a Host for Molecular Cloning in aStandard Cloning Pipeline

Recombinant DNA fragments for assembly were derived from multiplesources including, but not limited to: digestion of existing recombinantDNA using nucleases (e.g., restriction enzymes, homing endonucleases,zinc-finger nucleases, TALENs, Cas9 nuclease with appropriate guideRNAs, etc.), PCR amplification, or de novo gene assembly fromsynthesized oligonucleotides.

In vitro assembly was carried out with any number of standard DNAconstruction techniques or commercially available kits including, butnot limited to: ligation of DNA fragments using a suitable DNA ligaseand Gibson Assembly. Alternatively, in vivo assembly can be performed ina compatible host cell, such as, for example, E. coli or S. cerevisiaefollowed by isolated of the assembled product.

Once in vitro or in vivo assembly and isolation, if appropriate, iscomplete, V natriegens competent cells that have been prepared accordingto the conjugation or electroporation protocol were transformedaccording to the appropriate protocol in either Example 2 or Example 3.Cells were plated on agar plates containing the appropriate antibioticand incubated for several hours to overnight at 30-37° C. for coloniesto appear.

Colonies isolated from agar plates containing appropriate antibioticswere used to inoculate growth media containing the same antibiotic.Cells were grown for ˜3 hours to overnight at 30-37° C. Cells wereharvested by centrifugation, and DNA was then extracted via standardmethods (e.g., alkaline lysis techniques) or commercially available kits(e.g., QIAspin Miniprep Kit from Qiagen). Extracted DNA was analyzed bystandard methods.

Example 5 The Use of V. natriegens as a Host for Inducible ProteinExpression

A series of plasmids was designed for inducible protein expression ofgreen fluorescent protein (GFP). The plasmids were designed to contain:

-   -   1) one of three promoters (the IPTG-inducible trc promoter,        arabinose-inducible araBAD promoter, or the temperature        inducible λ pR promoter modulated by the temperature-sensitive        cI857 repressor.    -   2) one of two origins of replication (the p15a origin of        replication or the origin from plasmid pBR325).    -   3) a green fluorescent protein (GFP) under the control of the        inducible promoter to monitor expressions.    -   4) a transcriptional terminator following GFP (rrnB        transcriptional termination sequence).    -   5) a chloramphenicol resistance gene for antibiotic selection.

The functional elements and their source plasmids are listed in Table 2:

TABLE 2 Element Source Plasmid trc promoter pTrcHisA araBAD promoterpKD46 λ pR promoter/temperature- 705-cre sensitive cI857 repressor p15aorigin4 pACYC184 pBR325 origin pBR325 GFP gene synthesized from oligosrrnB transcriptional termination sequence pTrcHisA chloramphenical acyltransferase gene pCC1BAC

The maps for the six plasmids are shown in FIG. 1. The plasmids wereassembled in vitro using Gibson Assembly and electroporated into V.natriegens following the protocols described in Example 4.

Cultures of individual transformed colonies were grown overnight in LBmedia (10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl)supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14mM MgCl2) at 30° C. with agitation at 200 RPM. On the following day thecultures were used to inoculate fresh salt-supplemented LB media at aratio of 1:100 overnight culture:fresh media. The cultures were grown at30° C. with agitation until an OD600 of 0.5. Cultures were then inducedwith appropriate inducer (0.2% arabinose, 1 mM IPTG, or shiftingtemperature to 42° C., for the araBAD, trc, and pR promotersrespectively). After ˜4 hours, the OD600 and GFP fluorescence(excitation 480 nm/emission 510 nm) were measured. FIG. 2 shows GFPfluorescence normalized to OD600 for induced and non-induced culturesharboring each of the six expression plasmids. The data demonstrates thefunctionality of these inducible promoter systems in this species.

For further analysis, cultures of V. natriegens harboring pBR322-trc-GFP(induced and non-induced) were collected via centrifugation, resuspendedin lysis buffer (20 mM Tris pH 8, 2 mM MgCl₂), lysed via sonication,clarified via centrifugation, and run on a 4-12% 10-well Bolt® Bis-Trisgel (Life Technologies®) with MES running buffer, which was subsequentlystained with SimplyBlue™ safe stain (Life Technologies®). In FIG. 3,Lane 1: SeeBlue Plus2 Protein Standard (Life Technologies™); Lane 2: 1μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture; Lane3: 10 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture;Lane 4: 1 μL V. natriegens pBR322-trc-GFP lysate from non-inducedculture and Lane 5: 10 μL V. natriegens pBR322-trc-GFP lysate fromnon-induced culture. The GFP protein can be seen in the lanescorresponding to the induced culture, but is present at much lowerlevels in the non-induced culture.

Example 6 The Use of the “Free” ChrII as a Cloning/Shuttle Vector inNon-Vibrio Species (e.g., E. coli)

The replication machinery of V. natriegens chrII comprises SEQ ID NO: 1.

The vector pVnatoriCII was prepared by assembling the following DNAregions from the following sources:

1) V. natriegens chrII sequence (amplified from V. natriegens genomicDNA) (SEQ ID NO: 1);

2) R6Kγ origin of replication (amplified from plasmid pR6Kan fromEpicentre®)(SEQ ID NO: 2); and

3) tetA/tetR resistance genes+RP4 oriT (amplified from plasmid pJB3Tc20)(SEQ ID NO: 3).

The PCR primers were designed to generate sufficient homology overlapsbetween the PCR products to facilitate vector construction via GibsonAssembly to generate the plasmid shown in FIG. 4.

The plasmid comprises the sequence from V. natriegens chrII, the R6Kγorigin of replication (without the pir gene encoding the Π (pi) proteinnecessary for plasmid replication) and the tetA/tetR resistance genes(as a selective marker) along with the RP4 oriT region (to facilitatemobilization of plasmid via conjugation) of plasmid pJB3Tc20 (NCBIgenbank U75324).

The vector was assembled in vitro according to standard methods and wastransformed into EC100D pir-116 E. coli cells from Epicentre® viaelectroporation. These cells contain the pir gene encoding the Π (pi)protein necessary for replication of plasmids containing the R6Kγ originof replication. Because the designed plasmid contains the R6Kγ origin,the plasmid will be able to replicate in this strain regardless of thefunctionality of the V. natriegens chrII machinery in E. coli. Cellswere plated out on LB agar plates containing 10 μg/mL tetracycline.Individual colonies were grown up in LB media containing 10 μg/mLtetracycline, and DNA was recovered using the Qiaprep Spin Miniprep Kitfrom Qiagen®. Proper vector assembly was verified via restrictiondigestion analysis. Plasmids with the correct restriction pattern werethen electroporated into EPI300 E. coli cells from Epicentre® viaelectroporation. Because EPI300 cells do not contain the pir geneencoding then protein necessary for replication of plasmids containingthe R6Kγ origin of replication, the only way this plasmid will replicateis if the V. natriegens chrII replication machinery is able to supportplasmid replication in E. coli. The transformation of EPI300 cells withpVnatoriCII resulted in tetracycline resistant colonies, indicating theplasmid successfully replicated in this strain.

Conjugation Mediated Transfer of pVnatoriCII from E. coli to V.natriegens

The plasmid was also transferred to V. natriegens via conjugation fromE. coli strain 517-1 λpir following the conjugation protocol describedin Example 2, giving rise to tetracycline-resistant V. natriegenscolonies.

Cloning Large DNAs into pVnatoriCII

To assess the utility of pVnatoriCII for harboring large DNA moleculesin E. coli, we cloned ˜135 kb of sequence from the Mycoplasma genitaliumgenome into the plasmid (by replacing the R6Kγ origin with M. genitaliumsequence) to generate plasmid pVnoriCII-Mgen25-49. A plasmid of >100 kbcould be recovered from EPI300 cells harboring the plasmid as can beseen by running supercoiled plasmid DNA on an agarose gel (FIG. 5).

Sequencing of the plasmid (Illumina® MiSeq) confirmed the expectedsequence, demonstrating that pVnoriCII can be used to clone >100 kb ofexogenous DNA in E. coli.

Improved E. coli/V. natriegens Shuttle Vector

In order to improve upon the design of pVnatoriCII, a second V.natriegens chrII plasmid was designed (known aspVnatCII-YACTRP-copycontrol) by leveraging features from the followingplasmids:

From pVnoriCII:

-   -   oriT for RP4-mediated conjugal transfer.    -   V. natriegens chrII origin of replication for low copy        replication of plasmid.

From pCC1BAC™ from Epicentre®:

-   -   oriV for use with E. coli strains containing the trfA gene        product under an inducible promoter (e.g., EPI300 cells from        Epicentre®).    -   chloramphenicol resistance marker for antibiotic selection.    -   Multiple Cloning Site (MCS) with convenient restriction enzyme        cut sites.    -   loxP site for recombination via Cre-recombinase.

In addition, the plasmid also contains:

-   -   ARS/CEN for stabile replication in Saccharomyces cerevisiae.    -   Trp gene for selection in a Trp auxotroph of Saccharomyces        cerevisiae.

The vector map and sequence of pVnatCII-YACTRP-copy control are shown inFIG. 6.

The plasmid comprises the following sequence:pVnatCII-YACTRP-copycontrol (SEQ ID NO: 4).

The plasmid pVnatCII-YACTRP-copycontrol was replicated in E. coli at lowcopy, and the copy number was also increased by supplementing the trfAgene product in trans (e.g., via EPI300 cells, which contain aninducible copy of trfA on the chromosome).

The plasmid pVnatCII-YACTRP-copycontrol was also introduced into E.coli, V. natriegens, and Saccharomyces cerevisiae via transformation andmaintained under the appropriate selection.

Example 7 Use of a Suicide Plasmid System to Engineer the Genome of V.natriegens

We have developed a suicide plasmid system that can be used to removeendogenous DNA sequence or introduce exogenous DNA into the chromosomeof V natriegens. The plasmid was constructed with the following DNAelements in the following order:

-   -   a) 500 bp of chromosomal sequence directly upstream of the        location where an insertion/deletion event was desired to start;    -   b) A “knock-out/knock-in” cassette containing an antibiotic        resistance marker (e.g., Cm antibiotic resistance marker (from        pACYC184)) flanked by lox66 and lox71 sites. In addition, if        exogenous DNA was to be added into the chromosome, that DNA was        contained in this fragment after the lox-bounded Cm marker;    -   c) 500 bp of chromosomal sequence directly downstream of the        location where an insertion/deletion event was desired to end;    -   d) the R6K origin of replication from pR6Kan (Epicentre®);    -   e) the RK2 origin of transfer (oriT) from pRL443; and    -   f) the ccdB toxin under control of the arabinose-inducible        araBAD promoter (SEQ ID NO:5) (the araC gene and araBAD promoter        are from plasmid pKD46).

Because the plasmid lacks the Π (pi) replication protein necessary toinitiate replication from the R6K origin, the plasmid will onlyreplicate when Π (pi) is supplied in trans (e.g., from the EC100Dpir-116 strain from EPICENTRE®). The plasmid was introduced into an E.coli strain capable of supplying the Π (pi) protein in trans that alsocontained the conjugation machinery from plasmid RP4 (we use strainS17-1 λpir). The strain was then mated with V. natriegens (following theconjugation protocol described in Example 2) to allow mobilization ofthe plasmid from the donor E. coli strain to V natriegens. Because theplasmid is incapable of replicating in V. natriegens, the only way thatantibiotic-resistant clones were isolated was if the plasmid integratedinto the chromosome via the regions of the plasmid that are homologousto the V. natriegens genome. Double-crossover integration events wereselected for by growing the strain in media (e.g., LB) containing0.2-0.4% L-arabinose as well as an antibiotic (the antibiotic which iscontained in the cassette flanked by homology to the V. natriegensgenome). The presence of arabinose induced the araBAD promoter, therebyproducing the ccdB toxin and removing cells that had not undergoneintegration via double-crossover recombination from the population (thetoxin is not present in cells that have undergone double-crossoverrecombination). Surviving clones were screened for successfulintegration via standard methods.

Use of the System to Remove Endogenous DNA Sequence from the Chromosome

In this embodiment, the “knock-out/knock-in” cassette was composedsimply of an antibiotic resistance gene flanked by lox sites oriented inthe same direction (e.g., the orthogonal and uni-directional lox66/lox71pair). The “knock-out/knock-in” cassette was flanked on either side by500-750 bp of V. natriegens chromosomal sequence that was chosen suchthat the antibiotic cassette was flanked by 500-750 bp of sequenceimmediately upstream of the start point of the desired deletion, and500-750 bp immediately downstream of the end point for the desireddeletion. Upon successful integration via double-crossoverrecombination, the region of the genome to be deleted was replaced bythe antibiotic cassette flanked by lox sites. The antibiotic cassettewas later removed from the genome via the expression of Cre recombinase,which recombined the lox sites, thus looping the antibiotic cassette outof the chromosome (see discussion of engineering with Cre recombinase inExample 8). In some examples, we used this technique to remove the ORFfor the Dns exonuclease. In another example, we used this technique toremove a 28 kb region of Chromosome I from strain CCUG 16374 harboring aputative restriction-modification system (FIG. 7).

Use of the System to Introduce Exogenous DNA into the Chromosome

In this embodiment, the “knock-out/knock-in” cassette was composed of anantibiotic resistance gene (which may or may not be flanked by lox sitesoriented in the same direction) as well as additional exogenous DNA tobe added into the chromosome. The “knock-out/knock-in” cassette wasflanked on either side by 500 bp of V. natriegens chromosomal sequencethat was chosen such that the “knock-in” cassette is flanked by 500 bpof sequence immediately upstream of the start point of the desiredinsertion, and 500 bp immediately downstream of the end point for thedesired insertion. Upon successful integration via double-crossoverrecombination, the exogenous DNA along with the antibiotic marker wasinserted into the genome at the desired location. If the antibioticcassette is flanked by lox sites, it was later removed from the genomevia the expression of Cre recombinase, which recombined the lox sites,looping the antibiotic cassette out of the chromosome (see discussion ofengineering with Cre recombinase in Example 8). In some examples, weused this technique to introduce an inducible T7 RNA polymerase gene(SEQ ID NO:7) into the genome (see discussion of protein expression viaan inducible T7 RNA polymerase in Example 9).

Example 8 Use of Site-Specific Recombinases to Engineer the Genome of V.natriegens

The use of site specific recombinases along with their target sequenceswas used to carry out insertions and deletions in the chromosome of V.natriegens and could additionally be used to carry out inversions. Wehave demonstrated the use of the Cre-lox system to remove sequencespresent in the chromosome that have been flanked by lox sites.

In Example 7 (Use of a suicide plasmid system to engineer the genome ofV. natriegens), a chloramphenicol marker flanked by lox66 and lox71sites (that are oriented in the same direction) was introduced into thechromosome in such a manner as to replace the entire ORF for the Dnsnuclease. By expressing Cre recombinase, recombination between the loxsites resulted in the removal of the antibiotic marker from thechromosome, leaving behind a native loxP site (thus allowing us torecycle our antibiotic marker). To this end we designed the plasmidpACYCtetoriTCre, which contains:

-   -   a) the p15a origin of replication from plasmid pACYC184;    -   b) the tetracycline resistance cassette from plasmid pJB3Tc20;    -   c) the RK2 the RK2 origin of transfer (oriT) from pRL443;    -   d) the temperature-inducible Cre expression cassette from        plasmid 705-Cre (from Gene Bridges GmbH).

Introduction of the plasmid into the strain (carrying the lox siteflanked modification) via electroporation, followed by incubation at 37°C. (to induce expression of Cre recombinase) resulted in the desiredphenotype (i.e., a strain that had undergone Cre-mediated recombinationto remove the antibiotic marker).

In addition to carrying out deletions, this system can be used tointroduce novel DNA into a chromosome (via recombination with anexogenous circular DNA containing a lox site) and additionally oralternatively to invert regions of the chromosome (with properorientation of the lox site).

Analogous systems are envisioned which rely on other site-specificrecombinases or integrases (e.g., phiC31 integrase, bxb1 integrase,etc.).

Example 9 The Introduction of an Inducible T7 RNA Polymerase Gene intothe V. natriegens Chromosome and its Use in Recombinant ProteinExpression

Using the suicide plasmid system described in Example 7, we haveintroduced the gene for T7 RNA polymerase (SEQ ID NO:7) under thecontrol of either:

-   -   a) The arabinose-inducible araBAD promoter (SEQ ID NO:5) and        araC regulator protein (from E. coli); or    -   b) The IPTG-inducible lac operon regulatory elements and lad        regulator protein (SEQ ID NO:6) (from E. coli) into the        chromosome of V. natriegens. This system allows for inducible,        robust protein expression from a plasmid-borne gene under        control of the T7 promoter. We denote the arabinose-inducible        strain araBAD-T7, and the IPTG-inducible strain lacI-T7.

In conjunction with the strain, we designed a plasmid known aspET325Cm-YGFP which is based off of plasmid pET28a (Novagen) andcontains the YGFP fluorescent protein under the T7 promoter. The vectordiffers from pET28a primarily in that the Cm marker from pACYC184 isused instead of the kanamycin resistance gene.

The plasmid was introduced into V. natriegens araBAD-T7 and lacI-T7strains as well as the wild type (wt) parental strain viaelectroporation (described in Example 3). Strains harboring the plasmidwere grown up overnight in Brain Heart Infusion Broth+v2 salts with 12.5ug/mL chloraphenicol at 30° C. with shaking at 200 RPM (v2 salts meansthe media was supplemented with additional salts at the followingconcentrations: 204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl₂). The next day50 mL of either LB+v2 salt media or BHI+v2 salt media with 15 ug/mLchloramphenicol in a 250 mL baffled flask was inoculated with 1/100thvolume of overnight culture and incubated at 30° C. When the OD600 wasbetween 0.6 and 0.9 the cultures were induced with 1 mM IPTG (wt andlacI-t7 strains) or 1 mM IPTG+0.2% arabinose (araBAD-T7 strain). At 6.5hrs post induction, the cultures were retrieved and the cells wereharvested via centrifugation. The pellets were suspended in buffer (50mM Tris pH 7.4, 300 mM NaCl, 5 mM Imidazole) to a total volume of about7 mL. The cells were then imaged under white light (FIG. 8A), or under ablue light transilluminator with orange filter (FIG. 8B). As can be seenin FIG. 8, the wt strain, even when induced did not make any protein.Both versions of the T7 expression system (araBAD-T7 and lacI-T7)expressed YGFP. The pellets were lysed via sonication, clarified viacentrifugation, and the lysate was analyzed by SDS-PAGE (FIG. 9). Theoverexpressed YGFP construct is apparent in the lacI-T7 and araBAD-T7strains.

Many configurations of this system are envisioned. In some embodimentsthe RNA polymerase could reside on a plasmid and the gene that is to beoverexpressed could be cloned into any number of vectors under controlof the T7 promoter.

Analogous expression strains could be generated using otherconfigurations of chromosomally integrated or plasmid-borne inducibleRNA polymerases, relying on other RNA polymerases (e.g., SP6 RNApolymerase, etc.) or inducible promoters (e.g., other chemicallyinducible promoters, temperature inducible promoters, etc.).

Example 10 Secretion of Recombinant Proteins from Vibrio Sp.

This examples shows the secretion of recombinant proteins directly intothe growth media.

A V. natriegens was engineered by replacing the open reading frame (ORF)for the Dns nuclease with a cassette encoding the lad protein and the T7RNA polymerase protein (T7 RNAP) where the T7 RNAP protein gene has amodified or weakened ribosome binding site. Induction of T7 RNAP wascarried out by culturing in the presence of IPTG, which allowedinducible expression of genes under control of the T7 promoter. Aplasmid containing the lad gene and the gene for the levansucrase enzymecontaining its native secretion signal (SEQ ID NO: 9) under the controlof the T7 promoter (FIG. 10) was introduced into the strain. Anovernight culture was diluted into a minimal media (media is prepared bycombining 66 mL 10× phosphate/citric acid buffer (133 g/L KH₂PO₄, 40 g/L(NH4) H₂PO₄, 17 g/L citric acid, pH 6.3), 27.9 mL 70% glucose, 1.58 mLMgSO4.7H₂O (500 g/L stock), 45.6 mL 5 M NaCl, and 518.92 mL ddH2O, andthe pH is adjusted to 6.8) containing 1 mM IPTG (to induce expression ofthe levansucrase protein) and cultured for 5 hours at 37° C. After 5hours, cells were collected via centrifugation, and the growth media wasfiltered to remove additional cells and debris. Secreted proteins wereexamined by either running various volumes of the spent growth media onan SDS-PAGE gel, or by precipitating the proteins from the growth mediausing TCA, followed by resuspension in a suitable buffer and running onan SDS-PAGE gel, which showed expected bands for standards and adistinct band at the MW of levansucrase, increasing with concentration,while the rest of the gel was clear (FIG. 11). It was thus shows thatwhen IPTG and the plasmid are present, a protein with the expected sizeof levansucrase was present in the growth media in high abundance.Quantification of total protein via the Bradford assay shows that about100 mg/L of protein was secreted from the cells in the 5-hourfermentation (FIG. 11). Thus, IPTG-induced enzyme secretion in theorganism containing the plasmid of FIG. 10 is demonstrated.

Although the disclosure has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the disclosure. Accordingly,the disclosure is limited only by the following claims.

What is claimed is:
 1. A Vibrio natriegens organism comprising: arecombinant nucleic acid comprising a nucleotide sequence encoding a T7RNA polymerase and a recombinant regulatory element 5′ to the nucleotidesequence encoding the T7 RNA polymerase that regulates expression of theT7 RNA polymerase; wherein the recombinant nucleic acid further encodesa modified ribosome binding site 5′ to the nucleotide sequence encodingthe T7 RNA polymerase; and wherein the ribosome binding site comprisesthe nucleotide sequence of SEQ ID NO: 8, or the nucleotide sequence ofSEQ ID NO: 8, except for 1, 2, or 3 nucleotide substitutionmodification(s) and wherein the ribosome binding site regulatestranslation of the T7 RNA polymerase.
 2. The organism of claim 1,further comprising a heterologous nucleic acid sequence whereinexpression of the heterologous nucleic acid sequence is regulated by theT7 RNA polymerase.
 3. The organism of claim 1, wherein the recombinantregulatory element comprises.
 4. The organism of claim 3, wherein theinducible promoter, the ribosome binding site, and the nucleic acidencoding the T7 RNA polymerase are integrated into chromosome I of theorganism.
 5. The organism of claim 3, wherein the inducible promoter isinduced by arabinose.
 6. The organism of claim 3, wherein the induciblepromoter is inducible with IPTG.
 7. The organism of claim 6, furthercomprising a lacI repressor for repression of the inducible promoter. 8.The organism of claim 2, wherein the heterologous nucleic acid sequenceencodes a heterologous protein, and further encodes a signal sequencefor secretion of the heterologous protein.
 9. The organism of claim 1,further comprising a deletion of at least one gene encoding anextracellular nuclease.
 10. The organism of claim 9, wherein theextracellular nuclease is Dns.
 11. The organism of claim 1, having adoubling time of less than 15 minutes.
 12. The organism of claim 2,wherein the heterologous nucleic acid sequence is at least 10 kb insize.
 13. A method of producing a biomolecule comprising: a) contactingthe Vibrio natriegens organism of claim 1 with a vector comprising aheterologous nucleic acid encoding the biomolecule; b) growing theVibrio natriegens organism in a growth-conducive medium wherein theheterologous nucleic acid is expressed, thereby producing thebiomolecule; and c) isolating the biomolecule.
 14. The method of claim13, wherein the vector comprises the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having 90% or greater sequence identitythereto, wherein the vector is capable of supporting its replication inthe Vibrio natriegens organism to produce the biomolecule.
 15. Themethod of claim 14, wherein the heterologous nucleic acid is at least 10kb in size.
 16. The method of claim 15, wherein the vector furthercomprises an inducible promoter operably linked to the heterologousnucleic acid encoding the biomolecule.
 17. The method of claim 14,wherein the vector is contacted and introduced into the Vibrionatriegens organism by conjugation, chemical competence, naturalcompetence, or electroporation.
 18. The method of claim 16, wherein thebiomolecule is a protein or peptide.